EP3532633A2 - Compositions and methods for the production of compounds - Google Patents

Compositions and methods for the production of compounds

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Publication number
EP3532633A2
EP3532633A2 EP17865512.2A EP17865512A EP3532633A2 EP 3532633 A2 EP3532633 A2 EP 3532633A2 EP 17865512 A EP17865512 A EP 17865512A EP 3532633 A2 EP3532633 A2 EP 3532633A2
Authority
EP
European Patent Office
Prior art keywords
polyketide synthase
domain
seq
nucleic acid
lal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17865512.2A
Other languages
German (de)
French (fr)
Other versions
EP3532633A4 (en
Inventor
Daniel C. GRAY
Brian R. Bowman
Gregory L. Verdine
Mathew Edward SOWA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ginkgo Bioworks Inc
Original Assignee
Ginkgo Bioworks Inc
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Filing date
Publication date
Application filed by Ginkgo Bioworks Inc filed Critical Ginkgo Bioworks Inc
Publication of EP3532633A2 publication Critical patent/EP3532633A2/en
Publication of EP3532633A4 publication Critical patent/EP3532633A4/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Polyketide natural products are produced biosynthetically by polyketide synthases (PKSs), e.g., type I polyketide synthases, in conjunction with other tailoring enzymes.
  • PKSs polyketide synthases
  • Polyketide synthases (PKSs) are a family of large, multi-domain proteins whose catalytic functions are organized into modules to produce polyketides.
  • the basic functional unit of polyketide synthase clusters is the module, which encodes a 2- carbon extender unit, e.g., derived from malonyl-CoA.
  • the modules generally present in a polyketide synthase include i) a loading module; ii) extending modules; and iii) releasing modules.
  • the minimal domain architecture required for polyketide chain extension and elongation includes the ketosynthase (KS), acyl-transferase (AT) and the ACP (acyl-carrier protein) domains, and the specific chemistry of each module is encoded by the AT domain and by the presence of the ⁇ -ketone processing domains: ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains.
  • KR ketoreductase
  • DH dehydratase
  • ER enoylreductase
  • Combinatorial biosynthesis is a general strategy that has been employed to engineer polyketide synthase (PKS) gene clusters to produce novel drug candidates (Weissman and Leadlay, Nature Reviews Microbiology, 2005).
  • PKS polyketide synthase
  • these strategies have relied on engineering PKS domain deletions and/or domain swaps within a module or by swapping an entire module from another cluster to produce a chimeric cluster.
  • the problem with this approach is that protein engineering of the polyketide megasynthases via wholesale domain replacement, insertion, or deletion can perturb the "assembly line" architecture of the PKS, thus drastically reducing the amount of polyketide synthesized.
  • the disclosure provides composition and methods for domain-level PKS engineering by utilizing short protein sequences in ⁇ -ketone processing domains that control enzymatic activity, i.e., ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains (FIGS. 2A and 2B).
  • KR ketoreductase
  • DH dehydratase
  • ER enoylreductase domains
  • the putative dead domain sequences are grafted onto live domains to inactivate domain activity and alter the chemical structure of the polyketide encoded by the cluster.
  • Heterologous expression of the modified clusters in Streptomyces expression hosts then may be used to produce the novel compounds.
  • the approach may further be utilized by performing multiple domain-level engineering operations into one cluster to generate a combinatorial library of engineered molecules.
  • the disclosure provides an engineered polyketide synthase, wherein the polyketide synthase includes one or more modified domains having altered enzymatic activity relative to a reference polyketide synthase including unmodified domains, wherein the engineered polyketide synthase is capable of producing a polyketide when expressed under conditions suitable to allow expression of a compound by the engineered polyketide synthase.
  • the engineered polyketide synthase includes two or more modified domains having altered enzymatic activity.
  • At least one modified domain has decreased enzymatic activity (e.g., at least one modified domain is functionally inactive).
  • the modified domain is a ⁇ -ketone processing domain (e.g., a
  • ketoreductase a dehydratase, or an enoylreductase
  • the disclosure provides a polyketide synthase including:
  • At least one of the first domain and the second domain is a ⁇ -ketone processing domain (e.g., a ketoreductase, a dehydratase, or an enoylreductase).
  • the first domain and the second domain are both ⁇ -ketone processing domains.
  • At least one of the first domain and the second domain is a functionally inactive domain. In some embodiments, both of the first domain and the second domain are functionally inactive domains.
  • the polyketide synthase includes (c) a conserved region of a domain (e.g., a functionally inactive domain) of a third polyketide synthase or the conserved region of a second domain of the second polyketide synthase.
  • the polyketide synthase includes (d) a conserved region of a domain (e.g., a functionally inactive domain) of a fourth polyketide synthase, the conserved region of a second domain of the third polyketide synthase, or the conserved region of a third domain of the second polyketide synthase.
  • a conserved region of a domain e.g., a functionally inactive domain
  • the functionally inactive domain includes the amino acid sequence of the conserved region of any one of SEQ ID NO: 1 -9.
  • the ⁇ -ketone processing domain includes a portion having at least 90% sequence identity to the conserved region of any one of SEQ ID NO:1 -9.
  • the ⁇ -ketone processing domain is a ketoreductase, wherein the ketoreductase (a) includes an amino acid other than tyrosine at the position corresponding to the tyrosine in the conserved YAAAN catalytic motif and does not include the conserved aFG helix in SEQ ID NO:1 ; (b) includes a glutamic acid residue at the position
  • the ⁇ -ketone processing domain is a dehydratase, wherein the dehydratase includes (a) an aspartic acid at the position corresponding to the glycine at position 4288 in pksB of S679-pksB ORF in the conserved
  • the ⁇ -ketone processing domain is an enoylreductase, wherein the enoylreductase does not include a lysine at the position corresponding to position 1546 of S12-pksB ORF in SEQ ID NO: 8 and/or the aspartic acid at the position corresponding to position 1568 of S12-pksB in SEQ ID NO: 8 or 9.
  • the disclosure provides a chimeric polyketide synthase, wherein at least one domain of the polyketide synthase has been modified as compared to a polyketide synthase having the sequence of SEQ ID NO: 10 or 1 1 , wherein the modification results in altered enzymatic activity.
  • the disclosure provides, a chimeric polyketide synthase, wherein at least one ketoreductase domain (a) includes an amino acid other than tyrosine at the position corresponding to the tyrosine in the conserved YAAAN catalytic motif and does not include the conserved aFG helix in SEQ ID NO: 1 ; (b) includes a glutamic acid residue at the position corresponding to alanine 6632 of S9-pksA ORF in SEQ ID NO: 2; or (c) does not include the amino acids corresponding to amino acids 3386 to 3516 of WT S12-pksB ORF of SEQ ID NO: 3.
  • the disclosure provides a chimeric polyketide synthase, wherein at least one dehydratase domain (a) an aspartic acid at the position corresponding to the glycine at position 4288 in pksB of S679-pksB ORF in the conserved HXXXGXXXXP motif of SEQ ID NO: 4; (b) includes a substitution in the conserved LPFXW motif at the position corresponding to position 3066 to 3070 in S12- pksB ORF in SEQ ID NO: 5; (c) includes a deletion corresponding to positions between Pro 6844 and Trp 6874 of S679-pksA ORF of SEQ ID NO: 6; or (d) includes a substitution or deletion at the positions corresponding to A, B, C, and D of SEQ ID NO: 7.
  • the disclosure provides a chimeric polyketide synthase, wherein at least one enoylreductase domain does not include a lysine at the position corresponding to position 1546 of S12- pksB ORF in SEQ ID NO: 8 and/or the aspartic acid at the position corresponding to position 1 568 of S12-pksB in SEQ ID NO: 8 or 9.
  • the disclosure provides a chimeric polyketide synthase including a domain having at least 80% sequence identity to the amino acid sequence of (a) SEQ ID NO: 12, 13, or 14; (b) SEQ ID NO: 15, 1 6, or 17; (c) SEQ ID NO: 18, 19, or 20; (d) SEQ ID NO: 21 , 22, or 23; (e) SEQ ID NO: 24, 25, 26, or 27; (f) SEQ ID NO: 28, 29, 30, or 31 ; (g) SEQ ID NO: 32, 33, 34, or 35; or (h) SEQ ID NO: 36 or 37.
  • At least one enoyl reductase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 35-36.
  • the disclosure provides a nucleic acid encoding any of the foregoing polyketide synthases.
  • the nucleic acid further encodes an LAL, wherein the LAL includes a portion having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38.
  • the LAL includes a portion having the sequence of SEQ ID NO: 38.
  • the LAL has the sequence of SEQ ID NO: 38.
  • the nucleic acid lacks a TTA regulatory codon in at least one open reading frame.
  • the nucleic acid further includes an LAL binding site, e.g., an LAL binding site having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%) identity to the sequence of SEQ ID NO: 39 (CTAGGGGGTTGC).
  • the LAL binding site includes the sequence of SEQ ID NO: 39.
  • the LAL binding site has the sequence of SEQ ID NO: 39.
  • the LAL binding site includes the sequence SEQ ID NO: 40
  • the nucleic acid further includes an open reading frame positioned such that binding of the LAL to the LAL binding site promotes expression of the open reading frame.
  • the open reading frame encodes a compound-producing protein (e.g., a polyketide synthase).
  • the nucleic acid further encodes a nonribosomal peptide synthase. In some embodiments, the nucleic acid further encodes a first P450 enzyme. In some embodiments, the nucleic acid further encodes a second P450 enzyme.
  • the expression vector includes any of the foregoing nucleic acids.
  • the expression vector is an artificial chromosome (e.g., a bacterial artificial chromosome).
  • the disclosure provides a host cell including any of the foregoing vectors or polyketide synthases.
  • the polyketide synthase is heterologous to the host cell.
  • the host cell e.g., a host cell naturally lacking an LAL and/or an LAL binding site
  • a recombinant LAL e.g., a heterologous LAL
  • the LAL is constitutively active.
  • the host cell is engineered by insertion of a LAL binding site in a nucleic acid.
  • the binding of the recombinant LAL to the LAL binding site promotes transcription of the nucleic acid (e.g., a nucleic acid encoding a compound-producing protein such as a polyketide synthase).
  • the LAL binding site is heterologous to the LAL.
  • the LAL binding site is endogenous to the LAL.
  • the LAL binding site includes the sequence GGGGGT (SEQ ID NO: 40).
  • the host cell includes a nucleic acid including a heterologous LAL binding site operably linked to an open reading frame such that binding of an LAL to the heterologous LAL binding site promotes expression of the open reading frame.
  • the heterologous LAL binding site is a synthetic LAL binding site.
  • the heterologous LAL binding site promotes greater expression than the endogenous LAL binding site operably linked to the open reading frame.
  • the heterologous LAL binding site includes at least 8 contiguous nucleotides of C1 -T2-A3-G4-G5-G6-G7-G8-T9-T10-G11 -C12 (SEQ ID NO: 39), wherein none or up to six nucleotides other than any three nucleotides of G4, G5, G6, G7, Gs, T9, and T10 (e.g., G4, G7, and T9; G5, Gs, and T10; or G6, G7, and Gs) are replaced by any other nucleotide.
  • G4, G7, and T9 e.g., G4, G7, and T9; G5, Gs, and T10; or G6, G7, and Gs
  • the recombinant LAL includes a portion having at least 70% (e.g., at least
  • the recombinant LAL includes a portion having the sequence of SEQ ID NO: 38. In some embodiments, the recombinant LAL has the amino acid sequence of SEQ ID NO: 38.
  • the host cell is a bacterium (e.g., an actinobacterium such as
  • the actinobaceterium is S1391 , S1496, or S2441 .
  • the host cell has been modified to enhance expression of a compound-producing protein (e.g., a polyketide synthase).
  • a compound-producing protein e.g., a polyketide synthase
  • the host cell has been modified to enhance expression of a compound-producing protein (e.g., a polyketide synthase) by (i) deletion of an endogenous gene cluster which expresses a compound-producing protein (e.g., a polyketide synthase); (ii) insertion of a heterologous gene cluster which expresses a compound-producing protein (e.g., a polyketide synthase); (iii) exposure of the host cell to an antibiotic challenge; and/or (iv) introduction of a heterologous promoter that results in at least a two-fold increase in expression of a compound compared to the homologous promoter.
  • An additional method to enhance the expression of polyketides is to optimize media conditions for growth. This includes the specific chemical and nutri
  • the disclosure provides a method of producing a polyketide, the method including the step of culturing any of the foregoing host cells under suitable conditions.
  • the disclosure provides a method of producing a polyketide, the method including the step of culturing a host cell engineered to express any of the foregoing polyketide synthases under conditions suitable for the polyketide synthase to produce a polyketide.
  • the disclosure provides a method of modulating the activity of a polyketide synthase, the method including the steps of: (a) providing a parent nucleic acid sequence encoding a parent polyketide synthase; and (b) modifying at least one codon of the parent nucleic acid sequence, wherein the codon specifies a residue in a conserved motif of at least one domain of the parent polyketide synthase, wherein the modification results in an alteration of an enzymatic or regulatory activity (e.g., the alteration results in inactivity of the domain) of the at least one domain.
  • an enzymatic or regulatory activity e.g., the alteration results in inactivity of the domain
  • the disclosure provides a method of producing a compound, the method including the steps of: (a) providing a parent nucleic acid encoding a parent polyketide synthase; (b) modifying at least one codon (e.g., a codon in the portion of the nucleic acid which encodes a ⁇ -ketone processing domain) of the parent nucleic acid to create a modified nucleic acid encoding a modified polyketide synthase capable of producing a compound, wherein the codon specifies a residue in a conserved domain of at least one domain of the polyketide synthase and wherein the modification results in an alteration of the enzymatic activity of the at least one domain of the polyketide synthase; (c) introducing the modified nucleic acid to a host cell; and (d) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase, thereby producing a compound.
  • at least one codon e.g.,
  • the disclosure provides a method of producing a compound, the method including the steps of: (a) providing a parent polyketide synthase capable of producing a compound; (b) determining the amino acid sequence of the parent polyketide synthase; (c) providing a parent nucleic acid encoding the parent polyketide synthase; (d) modifying at least one codon of the parent nucleic acid to create a modified nucleic acid sequence encoding a modified polyketide synthase capable of producing a compound, wherein the codon specifies a residue in a conserved domain of at least one domain (e.g., a ⁇ -ketone processing domain) of the polyketide synthase and wherein the modification results in an alteration of the enzymatic activity (e.g., a decrease in activity) of the at least one domain; (e) introducing the modified nucleic acid to a host cell; (f) culturing the host cell under conditions suitable to allow expression of a compound by
  • the disclosure provides a method of producing a compound, the method of including the steps of: (a) determining the structure of a parent polyketide synthase; (b) producing a parent nucleic acid encoding the parent polyketide synthase; (c) modifying the nucleic acid to produce a modified nucleic acid encoding a modified polyketide synthase, wherein at least one domain (e.g., a ⁇ - ketone processing domain) of the modified polyketide synthase has altered enzymatic activity (e.g., decreased enzymatic activity) compared to the parent polyketide synthase; (d) introducing the modified nucleic acid sequence to a host cell; and (e) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase, thereby producing a compound.
  • a domain e.g., a ⁇ - ketone processing domain
  • the disclosure provides a method of producing a library of compounds, the method including the steps of: (a) providing a parent nucleic acid sequence encoding a parent polyketide synthase; (b) modifying at least one codon of the parent nucleic acid sequence to create a first modified nucleic acid encoding a first modified polyketide synthase capable of producing a compound; (c) modifying at least one codon of the parent nucleic acid to create a second modified nucleic acid encoding a second modified polyketide synthase capable of producing a compound, wherein the first and second modified nucleic acids are different; (d) introducing the first and the second modified nucleic acid sequences to one or more host cells; and (e) culturing the one or more host cells under conditions suitable to allow expression of a compound by the first and the second modified polyketide synthase, thereby producing a library of compounds.
  • the disclosure provides a compound produced by any of the foregoing methods.
  • conserved region of a domain refers to the portion of a domain of a polyketide synthase that is substantially the same in all domains of the same type which are active.
  • engineered polyketide synthase is used to describe a non-natural polyketide synthase whose design and/or production involves action of the hand of man.
  • an "engineered” polyketide synthase is prepared by production of a non-natural polynucleotide which encodes the polyketide synthase.
  • a cell that is "engineered to contain” and/or “engineered to express” refers to a cell that has been modified to contain and/or express a protein that does not naturally occur in the cell.
  • a cell may be engineered to contain a protein, e.g., by introducing a nucleic acid encoding the protein by introduction of a vector including the nucleic acid.
  • the term “functionally inactive,” as used herein, refers to a domain of a polyketide synthase that has no activity, or activity below the point of detection.
  • the term “gene cluster that produces a small molecule,” as used herein refers to a cluster of genes which encodes one or more compound-producing proteins.
  • heterologous refers to a relationship between two or more proteins, nucleic acids, compounds, and/or cell that is not present in nature.
  • the LAL having the sequence of SEQ ID NO: 38 is naturally occurring in the S1 8 Streptomyces strain and is thus homologous to that strain and would thus be heterologous to the S12 Streptomyces strain.
  • homologous refers to a relationship between two or more proteins, nucleic acids, compounds, and/or cells that is present naturally.
  • the LAL having the sequence of SEQ ID NO: 38 is naturally occurring in the S1 8 Streptomyces strain and is thus homologous to that strain.
  • modified domain refers to a domain of a polyketide synthase in which at least one amino acid residue has been altered from a reference sequence.
  • polyketide synthase refers to an enzyme belonging to the family of multi-domain enzymes capable of producing a polyketide.
  • a polyketide synthase may be expressed naturally in bacteria, fungi, plants, or animals.
  • recombinant refers to a protein that is produced using synthetic methods.
  • reference polyketide synthase refers to a polyketide synthase that has a sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) to the sequence of an engineered polyketide synthase except to the sequence of domains which are modified.
  • FIG. 1 is an image illustrating the biosynthesis of polyketides by a polyketide synthase.
  • FIGS. 2A and 2B are images illustrating modification of malonyl ⁇ -ketones by domains of polyketide synthases.
  • FIG. 3 is an image illustrating the biosynthesis of a polyketide.
  • FIGS. 4A and 4B are sequence alignments illustrating mutations resulting in inactive domains in polyketide synthases.
  • FIG. 5A is an image illustrating the deactivation of a ketoreductase domain.
  • FIGS. 5B and 5C are images illustrating the generation of a compound by a modified polyketide synthase.
  • FIGS. 6A-6C are images illustrating the generation of compounds by modified polyketide synthases.
  • FIG. 7A is an image of a compound generated by a modified polyketide synthase.
  • FIG. 7B is a graph illustrating generation of a compound by a modified polyketide synthase.
  • FIG. 7C is an image illustrating binding of a compound to CEP250.
  • FIG. 7D is a graph illustrating binding of compounds to CEP250.
  • FIG. 8A is an image illustrating a target-ID method used for assaying compounds in crude extracts.
  • FIG. 8B is an image illustrating binding of compounds to CEP250 and CBY1 .
  • FIG. 9A is an image illustrating deactivation of a ketoreductase domain in a polyketide synthase.
  • FIG. 9B is an image illustrating generation of a ring expanded compound by deactivation of a ketoreductase domain in a polyketide synthase.
  • ketoreductase KR
  • dehydratase DH
  • ER enoylreductase
  • Compounds that may be produced with the methods of the invention include, but are not limited to, polyketides and polyketide macrolide antibiotics such as erythromycin ; hybrid polyketides/non- ribosomal peptides such as rapamycin and FK506; carbohydrates including aminoglycoside antibiotics such as gentamicin, kanamycin, neomycin, tobramycin ; benzofuranoids; benzopyranoids; flavonoids; glycopeptides including vancomycin; lipopeptides including daptomycin; tannins; lignans; polycyclic aromatic natural products, terpenoids, steroids, sterols, oxazolidinones including linezolid; amino acids, peptides and peptide antibiotics including polymyxins, non-ribosomal peptides, ⁇ -lactams antibiotics including carbapenems, cephalosporins, and penicillin; purines, pteridines, polypyrroles,
  • Polyketide synthases are a family of multi-domain enzymes that produce polyketides.
  • Type I polyketide synthases are large, modular proteins which include several domains organized into modules.
  • the modules generally present in a polyketide synthase include i) a loading module; ii) extending modules; and iii) releasing and/or cyclization modules depending on whether the final polyketide is linear or cyclic.
  • the domains which generally are found in the modules are acyltransferase, acyl carrier protein, keto-synthase, ketoreductase, dehydratase, enoylreductase, methyltransferase, sulfhydrolase, and thioesterase.
  • a polyketide chain and the starter groups are generally bound to the thiol groups of the active site cysteines in the ketosynthase domain (the polyketide chain) and acyltransferase domain (the loading group and malonyl extender units) through a thioester linkage.
  • Binding to acyl carrier protein (ACP) is mediated by the thiol of the phosphopantetheinyl group, which is bound to a serine hydroxyl of ACP, to form a thioester linkage to the growing polyketide chain.
  • the growing polyketide chain is handed over from one thiol group to another by trans-acylations and is released after synthesis by hydrolysis or cyclization.
  • the synthesis of a polyketide begins by a starter unit, being loaded onto the acyl carrier protein domain of the PKS catalyzed by the acyltransferase in the loading module.
  • An extender unit e.g., a malonyl-CoA, is loaded onto the acyl carrier protein domain of the current module catalyzed by another acyltransferase domain.
  • the polyketide chain is then elongated by subsequent extender modules after being passed from the acyl carrier protein domain of module n to the ketosynthase domain of the n+1 module.
  • the acyl carrier protein bound extender unit reacts with the polyketide chain bound to the ketosynthase domain with expulsion of CO2 to produce an extended polyketide chain bound to the acyl carrier protein.
  • Each added extender unit may then be modified by ⁇ -ketoprocessing domains, i.e., ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons).
  • a thioesterase domain in the releasing modules hydrolyzes the completed polyketide chain from the acyl carrier protein of the last extending module.
  • the compound released from the PKS may then be further modified by other proteins, e.g., nonribosomal peptide synthase.
  • FIG. 3 An example of the synthesis of a polyketide by a PKS is illustrated in FIG. 3.
  • the biosynthetic cluster harbors polyketide megasynthases and a non-ribosomal peptide synthase (NRPS).
  • NRPS non-ribosomal peptide synthase
  • This hybrid architecture is referred to as hybrid PKS/NRPS.
  • the NRPS module inserts the pipecolate moiety in the FKBP12-binding region of the molecules (FIG. 3).
  • ⁇ -ketone processing domains are the domains in a PKS which result in modification of the elongation groups added during the synthesis of a polyketide. Each ⁇ -ketone processing domain is capable of changing the oxidation state of an elongation group.
  • the ⁇ -ketone processing domains include ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons).
  • KR3 from S9 includes single Ala to Glu substitution near the conserved catalytic YAAAN motif. While not being bound by the theory, homology modeling (using PDB 2FRO) suggested that a glutamic acid at this position might form a salt bridge with a nearby arginine, and that the resulting salt bridge would block the mobility of the substrate capping region (aFG) and prevent access of the ketoreductase active site to the polyketide substrate.
  • the S303 and S399 dead KR6 domains include more prominent lesions. In S303, the catalytic Tyr is replaced by Phe and the aFG helix was deleted. In S399, a larger 150 residue deletion encompassing the catalytic and substrate binding residues is present.
  • At least one ketoreductase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 12-23.
  • S679-DH7 For dehydratase domain-level engineering, four "dead" DH domains were analyzed: S679-DH7, S12-DH6, S12-DH7, S679-DH4, and S12-DH2.
  • the essential active site residues of the DH domain are distributed across four key conserved motifs: HXXXGXXXXP, GYXYGPXF, DXXX(Q/H) and LPFXW.
  • S679-DH7 has a single Gly to Asp substitution in the HXXXGXXXXP motif, which contains the His residue that deprotonates the polyketide substrate to initiate the dehydration reaction.
  • S12-DH6 and S12- DH7 have substitutions in the LPFXW motif.
  • S679-DH4 contains a significant internal deletion, and S12- DH2 harbors mutations in all four key motifs comprising the DH active site.
  • Hybrid PKS/NRPS clusters such as rapamycin require a hydroxyl for macrocyclization, and thus the S12-DH2 "dead" DH domain must remain inactive for cyclization and biological activity.
  • At least one dehydratase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 24-35.
  • At least one enoyl reductase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to SEQ ID No: 36 or 37.
  • LALs include three domains, a nucleotide-binding domain, an inducer-binding domain, and a DNA-binding domain.
  • a defining characteristic of the structural class of regulatory proteins that include the LALs is the presence of the AAA+ ATPase domain.
  • Nucleotide hydrolysis is coupled to large conformational changes in the proteins and/or multimerization, and nucleotide binding and hydrolysis represents a "molecular timer" that controls the activity of the LAL (e.g., the duration of the activity of the LAL).
  • the LAL is activated by binding of a small-molecule ligand to the inducer binding site. In most cases the allosteric inducer of the LAL is unknown.
  • the allosteric inducer is maltotriose.
  • Possible inducers for LAL proteins include small molecules found in the environment that trigger compound (e.g., polyketide) biosynthesis.
  • the regulation of the LAL controls production of compound-producing proteins (e.g., polyketide synthases) resulting in activation of compound (e.g., polyketide) production in the presence of external environmental stimuli.
  • the LAL is a fusion protein.
  • an LAL may be modified to include a non-LAL DNA-binding domain, thereby forming a fusion protein including an LAL nucleotide-binding domain and a non-LAL DNA-binding domain.
  • the non-LAL DNA-binding domain is capable of binding to a promoter including a protein-binding site positioned such that binding of the DNA-binding domain to the protein- binding site of the promoter promotes expression of a gene of interest (e.g., a gene encoding a compound-producing protein, as described herein).
  • the non-LAL DNA binding domain may include any DNA binding domain known in the art. In some instances, the non-LAL DNA binding domain is a transcription factor DNA binding domain.
  • non-LAL DNA binding domains include, without limitation, a basic helix-loop-helix (bHLH) domain, leucine zipper domain (e.g., a basic leucine zipper domain), GCC box domain, helix-turn-helix domain, homeodomain, srf-like domain, paired box domain, winged helix domain, zinc finger domain, HMG-box domain, Wor3 domain, OB-fold domain,
  • bHLH basic helix-loop-helix
  • leucine zipper domain e.g., a basic leucine zipper domain
  • GCC box domain e.g., a basic leucine zipper domain
  • helix-turn-helix domain e.g., a basic leucine zipper domain
  • homeodomain e.g., a basic leucine zipper domain
  • srf-like domain e.g., a basic leucine zipper domain
  • paired box domain e.g., paired box domain
  • the promoter is positioned upstream to the gene of interest, such that the fusion protein may bind to the promoter and induce or inhibit expression of the gene of interest.
  • the promoter is a heterologous promoter introduced to the nucleic acid (e.g., a chromosome, plasmid, fosmid, or any other nucleic acid construct known in the art) containing the gene of interest.
  • the promoter is a preexisting promoter positioned upstream to the gene of interest.
  • the protein-binding site within the promoter may, for example, be a non-LAL protein-binding site.
  • the protein- binding site binds to the non-LAL DNA binding domain, thereby forming a cognate DNA binding domain/ protein-binding site pair.
  • the LAL is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 41 -62 or has a sequences with at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 63-73.
  • 70% e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%
  • a gene cluster (e.g., a PKS gene cluster) includes one or more promoters that include one or more LAL binding sites.
  • the LAL binding sites may include a polynucleotide consensus LAL binding site sequence (e.g., as described herein).
  • the LAL binding site includes a core AGGGGG (SEQ ID NO: 74) motif.
  • the LAL binding site includes a sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) homology to SEQ ID NO: 39.
  • the LAL binding site may include mutation sites that have been restored to match the sequence of a consensus or optimized LAL binding site.
  • the LAL binding site is a synthetic LAL binding site.
  • synthetic LAL binding sites may be identified by (a) providing a plurality of synthetic nucleic acids including at least eight nucleotides; (b) contacting one or more of the plurality of nucleotides including at least eight nucleotides with one or more LALs; (c) determining the binding affinity between a nucleic acid of step (a) and an LAL of step (b), wherein a synthetic nucleic acid is identified as a synthetic LAL binding site if the affinity between the synthetic nucleic acid and an LAL is greater than X.
  • the identified synthetic LAL binding sites may then be introduced into a host cell in a compound-producing cluster (e.g., a PKS cluster).
  • a pair of LAL binding site and a heterologous LAL or a heterologous LAL binding site and an LAL that have increased expression compared to a natural pair may be identified by (a) providing one or more LAL binding sites; (b) contacting one or more of the LAL binding sites with one or more LALs; (c) determining the binding affinity between a LAL binding site and an LAL, wherein a pair having increased expression is identified if the affinity between the LAL binding site and the LAL is greater than the affinity between the LAL binding site and its homologous LAL and/or the LAL at its homologous LAL binding site.
  • the binding affinity between the LAL binding site and the LAL is determined by determining the expression of a protein or compound by a cell which includes both the LAL and the LAL binding site.
  • the recombinant LAL is a constitutively active LAL.
  • the amino acid sequence of the LAL has been modified in such a way that it does not require the presence of an inducer compound for the altered LAL to engage its cognate binding site and activate transcription of a compound producing protein (e.g., polyketide synthase).
  • a constitutively active LAL to a host cell would likely result in increased expression of the compound-producing protein (e.g., polyketide synthase) and, in turn, increased production of the corresponding compound (e.g., polyketide).
  • FkPhD gene clusters are arranged with a multicistronic architecture driven by multiple bidirectional promoter-operators that harbor conserved (in single or multiple, and inverted to each other and/or directly repeating) GGGGGT (SEQ ID NO: 40) motifs presumed to be LAL binding sites.
  • Bidirectional LAL promoters may be converted to unidirectional ones (UniLALs) by strategically deleting one of the opposing promoters, but maintaining the tandem LAL binding sites (in case binding of LALs in the native promoter is cooperative, as was demonstrated for MalT). Functionally this is achieved by removal of all sequences 3' of the conserved GGGGGT (SEQ ID NO: 40) motif present on the antisense strand (likely containing the -35 and -10 promoter sequences), but leaving intact the entire sequence on the sense strand. As a consequence of this deletion, transcription would be activated in one direction only.
  • the advantages of this feed-forward circuit architecture would be to tune and/or maximize LAL expression during the complex life cycle of Streptomyces vegetative and fermentation growth conditions.
  • the host cell is a bacteria such as an Actiobacterium.
  • the host cell is a Streptomyces strain.
  • the host cell is Streptomyces anulatus, Streptomyces antibioticus, Streptomyces coelicolor, Streptomyces peucetius, Streptomyces sp.
  • Streptomyces canus Streptomyces nodosus, Streptomyces (multiple sp.), Streptoalloteicus hindustanus, Streptomyces hygroscopicus, Streptomyces avermitilis, Streptomyces viridochromogenes, Streptomyces verticillus, Streptomyces chartruensis, Streptomyces (multiple sp.), Saccharothrix mutabilis, Streptomyces halstedii, Streptomyces clavuligerus, Streptomyces venezuelae, Strteptomyces roseochromogenes, Amycolatopsis orientalis, Streptomyces clavuligerus, Streptomyces rishiriensis, Streptomyces lavendulae, Streptomyces roseosporus, Nonomuraea sp., Streptomyces peucetius
  • Streptomyces aureofaciens Streptomyces natalensis, Streptomyces chattanoogensis L 10, Streptomyces lydicus A02, Streptomyces fradiae, Streptomyces ambofaciens, Streptomyces tendae, Streptomyces noursei, Streptomyces avermitilis, Streptomyces rimosus, Streptomyces wedmorensis, Streptomyces cacaoi, Streptomyces pristinaespiralis, Streptomyces pristinaespiralis, Actinoplanes sp. ATCC 33076, Streptomyces hygroscopicus, Lechevalieria aerocolonegenes, Amycolatopsis mediterranei,
  • Amycolatopsis lurida Streptomyces albus, Streptomyces griseolus, Streptomyces spectabilis
  • Streptomyces ambofaciens Streptomyces staurosporeus, Streptomyces griseus, Streptomyces (multiple species), Streptomyces acromogenes, Streptomyces tsukubaensis, Actinoplanes teichomyceticus, Streptomyces glaucescens, Streptomyces rimosus, Streptomyces cattleya, Streptomyces azureus, Streptoalloteicus hindustanus, Streptomyces chartreusis, Streptomyces fradiae, Streptomyces coelicolor, Streptomyces hygroscopicus, Streptomyces sp.
  • the host cell is an Escherichia strain such as Escherichia coli.
  • the host cell is a Bacillus strain such as Bacillus subtilis.
  • the host cell is a Pseudomonas strain such as Pseudomonas putitda.
  • the host cell is a Myxococcus strain such as Myxococcus xanthus.
  • the proteins, nucleic acids, vectors, and host cells of the invention may be used for production of compounds (e.g., polyketides). Introduction of heterologous domains to proteins allow alteration of the chemical structure of polyketides produced by the proteins.
  • the activity of ⁇ -ketone processing domains can be altered by introducing the sequences of domains from other polyketide synthases. Multiple heterologous sequences can be tested for their ability to alter the activity of a specific domain without drastically reducing the amount of polyketide expressed. New variants of the polyketide synthase can be subjected to rigorous quality control (Sanger sequencing of region of interest, PCR-based "tiling" to confirm cluster integrity and lllumina sequencing to sequence the entire BAC). BACs may then be conjugated to two optimized Streptomyces producer strains, and solid-phase extracted (SPE) samples can be subjected to Top-Down mass spectrometry with purified FKBP12 protein to identify produced compounds.
  • SPE solid-phase extracted
  • a representative example of a workflow to generate a compound from a chimeric polyketide synthase includes grafting a short peptide sequence from the domain of one polyketide synthase, e.g., a ketoreductase domain, onto another polyketide synthase using homology-based cloning.
  • the catalytic Tyr of one ketoreductase may be replaced with Phe and the active site aFG loop may also deleted to inactive the domain.
  • the resulting clone may then be conjugated into a Streptomyces expression host and fermented.
  • Compounds may then be identified using comparative LC-TOF analysis of unfractionated SPE samples.
  • Top Down mass spectrometry analysis may also be performed by co- injecting purified native FKBP12 and a compound from the modified polyketide synthase with a compound from the unmodified polyketide synthase. This analysis can show a mass difference between the two compounds consistent with the change in activity of the domain, e.g., a difference of 2 for an inactivated ketoreductase domain.
  • Compounds with multiple structural changes may be generated using combinations of KR, DH or ER single variants.
  • Combinatorial domain level engineering may be performed by combining multiple domain-level variants on a single protein backbone, thus enabling library-scale construction of diverse PKS/NRPS molecules for drug development.
  • multiplex parallel engineering may be used to produce libraries of engineered PKS/NRPS molecules for drug development.
  • site-directed mutagenesis of a polynucleotide encoding a parent polyketide synthase may be used to generate, in parallel, a plurality of polynucleotides encoding a plurality of engineered polyketide synthases.
  • each of the plurality of engineered polyketide synthases includes at least one codon modification relative to of the parent polyketide synthase (e.g., a codon that specifies a residue in a conserved motif of at least one domain of the parent polyketide synthase).
  • single-molecule long-read sequencing technology e.g., Nanopore sequencing or SMRT sequencing
  • Single-molecule long-read sequencing may be used to characterize libraries of engineered polyketide synthases or non-ribosomal peptide synthases which are produced by any of the methods described herein.
  • single-molecule long-read sequencing e.g., Nanopore sequencing or
  • SMRT sequencing may be used to characterize (e.g., deconvolute) combinatorial or multiplex libraries of engineered polyketide synthases or non-ribosomal peptide synthases (e.g., multiplex libraries generated by parallel engineering).
  • Single-molecule long-read sequencing enables the identification of the module or modules which are incorporated into the combinatorial library. This further enables the prediction of the chemistry of the resulting plurality of engineered polyketide synthases or non-ribosomal peptide synthases. The predicted enzymatic chemistry can therefore be connected to the compounds produced by the engineered polyketide synthases or non-ribosomal peptide synthases.
  • the resulting compounds may be identified by chemical methods of analysis known to one of skill in the art (e.g., mass
  • the predicted enzymatic chemistry can be connected to the function of the resulting compounds (e.g., binding to a target protein or inducing a phenotype, such as a cell based phenotype). Accordingly, long-read sequencing of a genetically encoded molecule may allow for genotypic-phenotypic linkage.
  • Single-molecule long-read sequencing technologies may be considered to include any sequencing technology which enables the sequencing of a single molecule of a biopolymer (e.g., a polynucleotide such as DNA or RNA), and which enables read lengths of greater than 2 kilobases (e.g., greater than 5 kilobases, greater than 10 kilobases, greater than 20 kilobases, greater than 50 kilobases, or greater 100 kilobases).
  • Single-molecule long-read sequencing technologies may enable the sequencing of multiple single molecules of DNA or RNA in parallel.
  • Single-molecule long-read sequencing technologies may include sequencing technologies that rely on individual
  • Nanopore sequencing is an exemplary single-molecule long-read sequencing technology that may be used to characterize libraries of engineered polyketide synthases or non-ribosomal peptide synthases that are prepared by any of the methods described herein.
  • Nanopore sequencing enables the long-read sequencing of single molecules of biopolymers (e.g., polynucleotides such as DNA or RNA).
  • Nanopore sequencing relies on protein nanopores set in an electrically resistant polymer membrane. An ionic current is passed through the nanopores by setting a voltage across this membrane. If an analyte (e.g., a biopolymer such as DNA or RNA) passes through the pore or near its aperture, this event creates a characteristic disruption in current.
  • the magnitude of the electric current density across a nanopore surface depends on the composition of DNA or RNA (e.g., the specific base) that is occupying the nanopore. Therefore, measurement of the current makes it possible to identify the sequence of the molecule in question.
  • SMRT Single molecule real-time sequencing
  • PacBio is an exemplary single-molecule long- read sequencing technology that may be used to characterize libraries of engineered polyketide synthases or non-ribosomal peptide synthases that are prepared by any of the methods described herein.
  • SMRT is a parallelized single molecule DNA sequencing method.
  • SMRT utilizes a zero-mode waveguide (ZMW).
  • ZMW zero-mode waveguide
  • a single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template.
  • the ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase.
  • Each of the four DNA bases is attached to one of four different fluorescent dyes.
  • the fluorescent tag When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable.
  • a detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
  • a short peptide sequence from S303-KR6 was grafted onto X1 -KR6 using homology-based cloning.
  • the catalytic Tyr is replaced with Phe (shown in red) and the active site aFG loop (show in blue) is also deleted (FIG. 5A).
  • the resulting clone was conjugated into a Streptomyces expression host and fermented.
  • Comparative LC-TOF analysis of unfractionated SPE samples of Compound 1 and C16-keto- Compound 1 indicated that the new compound had the desired M+H mass of 608.35 (FIG. 5B).
  • FIG. 6B is a diagrammatic representation of FIG. 6B.
  • the rapamycin/FK506 "constant region" is the conserved portion of the macrolide ring that binds
  • FKBP12. DH8 in the PKS which produces Compound 1 was inactivated by mutating the LPFXW motif to generate Compound 2 with a hydroxyl in the pyran ring of the constant region (FIG. 7A).
  • the expected mass of 61 1 .38 (FIG. 7B) was observed by a Top-Down assay, which confirmed that Compound 2 retained FKBP12 binding affinity.
  • the structure of the FKBP12: Compound 2:CEP250 complex (FIG. 7C) was solved by crystallization.
  • Target-ID assay based on FKBP12 affinity enrichment and LC-MS/MS sequencing of tryptic peptides that allows for the identification of the protein targets of compounds in crude extracts was developed (FIG. 8A).
  • Target-ID analysis confirmed that Compound 1 bound both CEP250 and CBY1 Ain 293T lysates, whereas Compound 3, a combinatorial compound derivative of Compound 1 , selectively bound CBY1 and not CEP250.
  • the mass spectrometry-based Target-ID results were validated with TR-FRET data.
  • the TR-FRET assay confirmed that CBY1 binds to Compound 1 (FIG. 8B).
  • the data also confirmed that Compound 3 is specific for CBY1 and can no longer engage CEP250.
  • Compound 2 (FIG. 7A) is specific for CEP250 and not CBY1 .
  • the structure of Compound 3 was confirmed by NMR, which indicated the successful inactivation of KR6, DH4 and ER5 domains via the domain-level engineering approach described above.
  • Compound 2 also lacked the third carbonyl in the constant region, suggesting that CypB, the final tailoring step in Compound 1 biosynthesis, was unable to utilize Compound 2 as a productive substrate.
  • the above data demonstrates that domain-level engineering results in compounds with
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any polynucleotide or protein encoded thereby; any method of production; any method of use) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

Abstract

The present disclosure provides proteins, nucleic acids, vectors, and host molecules useful for the production of compounds of interest, and methods for their use.

Description

COMPOSITIONS AND METHODS FOR THE PRODUCTION OF COMPOUNDS
Background
Polyketide natural products are produced biosynthetically by polyketide synthases (PKSs), e.g., type I polyketide synthases, in conjunction with other tailoring enzymes. Polyketide synthases (PKSs) are a family of large, multi-domain proteins whose catalytic functions are organized into modules to produce polyketides. The basic functional unit of polyketide synthase clusters is the module, which encodes a 2- carbon extender unit, e.g., derived from malonyl-CoA. The modules generally present in a polyketide synthase include i) a loading module; ii) extending modules; and iii) releasing modules. Within the module, the minimal domain architecture required for polyketide chain extension and elongation includes the ketosynthase (KS), acyl-transferase (AT) and the ACP (acyl-carrier protein) domains, and the specific chemistry of each module is encoded by the AT domain and by the presence of the β-ketone processing domains: ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains. Polyketide synthase biosynthesis proceeds by two key mechanisms: polyketide chain elongation with a polyketide synthase extending module and translocation of the polyketide intermediate between modules.
Productive chain elongation depends on the concerted function of the numerous catalytic domains both within and between modules.
Combinatorial biosynthesis is a general strategy that has been employed to engineer polyketide synthase (PKS) gene clusters to produce novel drug candidates (Weissman and Leadlay, Nature Reviews Microbiology, 2005). To date, these strategies have relied on engineering PKS domain deletions and/or domain swaps within a module or by swapping an entire module from another cluster to produce a chimeric cluster. The problem with this approach is that protein engineering of the polyketide megasynthases via wholesale domain replacement, insertion, or deletion can perturb the "assembly line" architecture of the PKS, thus drastically reducing the amount of polyketide synthesized.
Summary of the Invention
The present disclosure provides compositions and methods useful to facilitate combinatorial biosynthesis of polyketides without a significant loss of compound production by mimicking and accelerating the mechanism by which domain activity is turned "on" or "off" by evolution (FIG. 1 ).
More specifically, the disclosure provides composition and methods for domain-level PKS engineering by utilizing short protein sequences in β-ketone processing domains that control enzymatic activity, i.e., ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains (FIGS. 2A and 2B). The putative dead domain sequences are grafted onto live domains to inactivate domain activity and alter the chemical structure of the polyketide encoded by the cluster. Heterologous expression of the modified clusters in Streptomyces expression hosts then may be used to produce the novel compounds. The approach may further be utilized by performing multiple domain-level engineering operations into one cluster to generate a combinatorial library of engineered molecules.
Accordingly, in one aspect, the disclosure provides an engineered polyketide synthase, wherein the polyketide synthase includes one or more modified domains having altered enzymatic activity relative to a reference polyketide synthase including unmodified domains, wherein the engineered polyketide synthase is capable of producing a polyketide when expressed under conditions suitable to allow expression of a compound by the engineered polyketide synthase.
In some embodiments, the engineered polyketide synthase includes two or more modified domains having altered enzymatic activity.
In some embodiments, at least one modified domain has decreased enzymatic activity (e.g., at least one modified domain is functionally inactive).
In some embodiments, the modified domain is a β-ketone processing domain (e.g., a
ketoreductase, a dehydratase, or an enoylreductase).
In another aspect, the disclosure provides a polyketide synthase including:
(a) a first domain including a conserved region of a domain of a first polyketide synthase; and
(b) a second domain including a conserved region of a domain of a second polyketide synthase.
In some embodiments, at least one of the first domain and the second domain is a β-ketone processing domain (e.g., a ketoreductase, a dehydratase, or an enoylreductase). In some embodiments, the first domain and the second domain are both β-ketone processing domains.
In some embodiments, at least one of the first domain and the second domain is a functionally inactive domain. In some embodiments, both of the first domain and the second domain are functionally inactive domains.
In some embodiments, the polyketide synthase includes (c) a conserved region of a domain (e.g., a functionally inactive domain) of a third polyketide synthase or the conserved region of a second domain of the second polyketide synthase.
In some embodiments, the polyketide synthase includes (d) a conserved region of a domain (e.g., a functionally inactive domain) of a fourth polyketide synthase, the conserved region of a second domain of the third polyketide synthase, or the conserved region of a third domain of the second polyketide synthase.
In some embodiments, the functionally inactive domain includes the amino acid sequence of the conserved region of any one of SEQ ID NO: 1 -9.
In some embodiments of any of the foregoing polyketide synthases, the β-ketone processing domain includes a portion having at least 90% sequence identity to the conserved region of any one of SEQ ID NO:1 -9.
SEQ ID NO: 1
DPDGTVLITGGSGVRAGALARHLVTERGVRHLLLLSRTTADEELLNELGELGARVDTAICDVSDRARLAQ VLAGVSPEHPLTAVIHTAGALDDDVVESLTAQRLDTVLRPKADGAWHLHELTRDTDLAAFVMYSSAAGV MGNPGQGNFAAATAFLDALAEQRRAEGLPALALAWGSSEETGGLTGLRAISAEHGMRLFDSASHRREPL LVAASMDPVLAAEVPALLRSLRRPIARRAASADGVQWLAGLAPEERAKALLKVVCDTAATVLGHADARTI PLTGAFKDLGVDSLTAVELRNSLTKATGLRLPATLVFDYPTPTALAVRL
SEQ ID NO: 2
DPDGTILITGGSGVLAGILARHLAAEHGARHLLLLSRTAPDEALIKELAELGARVETAACDVSDRAGLARVL AGVSPEHPLTAVIHTAGALDDGVVESLTTQQLDTVLRPKADGAWHLHELTRDADLAAFVVYSSAAAVLGN EGQGNYAAANAFLDALAEQRRTQGLPALALAWGPWEYTGDLTAQLTGTDQDRIRCSGMRTITAEDGMR LFDTASHHGEPLLVPAVLDPTRDGEVPALLRSLRRPIARRAASADGGVQWLAALAPAEREKALLKLVCDS AAMVLGHADARSIPAAGAFKDLGVDSLMAVELRNGLVKATGLRLPATLVFDYPTPTVLAARL
SEQ ID NO: 3
DPDGTVLITGERAGAVARRMAERGVRHLLLASGRVPDELMDLDTSVEVAVCDVSDRAALAGVLAGLPSL TGVIQTAGEDVLPVLAGAITPTRDGEIPASLRLLRRPLVRRRVSAAGDSSLAALPPAERERALLKVVRDSA AVVLGHADGRTVPATAAFKDLGLDSLTAVELRNSLRKATGLQLPATLVFDYPSPVALAARLG
SEQ ID NO: 4
HPFLGAALPAPDGDSLTLTGRITLDAHPWLADHIIRDTLILPGAAFAECVLRAGREVGCDLLEELVIEAPLVL PATGGVAVRIAVGEPDDAGRRTFDLYARPDAAPGWNRHAGGTLKPGDALPATEAATETVAWPPADAEP VDVDDLYDRLAAAGYAYGPAFQSVHAAWRTPDAIWAEVVLDGEPAGFGLHPALLDGALQLSALAATGGD VAQLPFAWHDVRLPGHGADRLRVRL SEQ ID NO: 5
HPLLGAIVAVPQSGGVAMTSRLSPRNHPWLAEHTLGGVPTVPTSVLVELAVRAGDEVGCGVVEELTVDA PLLLPERGGVRVQVIVGATDANGQRGLDIFSAPEDTGQEAWTRHATGTLAPGGDIAADVDLSAWPPANA QPVDVTDGYDLLERAGYGYGPAFQGVRAIWRRGEELFAEVALEPELTDTAARFGLHPALLDAAWHPELR DEVAETSPDGRRWWSQPSRWAGLRLHTAGATVLRVRLAPVDADSMSLQAADETGDPVLTVDSLS
SEQ ID NO: 6
HPLLGAGMPIAGTGAVLFGTEVAHPWFDGHETLPAAAFAEIAVRAAAEVGSPVVGELHVELLPRIPADGR
LRLQTWVDGPDPTGVRRFTVHARPDPTAAWLRVASGVLTGAEAPVPAFAGGEPLHIADGTPAGFLLHPD
ATPAADWFGLVAHGSGARQQHVYQAGEGLCVTDDAGRPIVTAARVR
SEQ ID NO: 7
HPLLGWGVPVAEAGGRLYTGRVARQDGPVLSVAAFVEMAFAAAGGRPIRELSVDALLYIPDDGTAELQT WVSEHRLTIHARYRDTEPWTRLATAALDTTAPATTHTPHPGLITTALTLTGDEAPAIWHDLTLHTSNATEL HTHITPGDDGTLTITATDTTGQPVLTAHTAT
SEQ ID NO: 8
RLSALASLGEPQIVVRDDTPLVARLAREKSPALTIPGERAWVLEPDHSGVLQELALVAADTDVRPLRPGE VRIEVRAAGLNFRDVLVALGTDLGDGVFGAEGAGVVLETGSDVRDLRPGDRVFGLLEGGHGSIAIADRR MLAVIPEGWSFATAASVPEVFVIAYYGLVDLAGLRAGESVLIHAATGGVGMAATQIARHLGAQVYATAGV GKQHILRDAGLGDDRIADSRTTDFREAFRDSTQGRGVDVVLNSLKGDFVDASLDLLADGGRFLELGQTDI RDAGEIAAERPGTTYHSFTRMNAGPDRLREIIAELLALFEQGVLRPSPVHTWDIRHAREAFSWMSGGRHT GKMVLTMPQRIDPGGTVLIAGDSEALARIAARHLGVRHLLLDRGVADAAPDAVVCDVSDHDALERVLADL SPEHPLTAVIHTGGAAVTDEIRRLHDLTESLDLTDFVVFSQDAPAAVEAFARSRRAHGLPVRTIAWGIPEA DPVVADEHLLGRALASAEQAQIVARVNTAGLRALTAANALPTLLRNLIRAEPEETGQSAWPHRFEAAGAD REEALLDLIRANVVDILSLPTADRYAPDRTFREMGIDSLTAVGLRNSLAKATGLPLPTTMVFDYPTPAVLTA RMREL SEQ ID NO: 9
RLSTLVALGEPQIALRDSTPLVPRLAPESSTALTTPAARAWVLEPARSGTLRELSLVAADTDARPLRPGEV RVDVRAAGLNFRDVLIALGTYPGDGVMGGEAAGVVLEVGPEVNDLSVGDRVFGLVTDGFGPVTITDRRL LAAMPQDWSFTTAASAAMAFATAHYGLVELAGLKAGESVLIHAATGGVGMAATQIAHHLGAHIYATASSG KQHLLRAAGIDDDRIANSRTTGFRDAFLDSTGGRGVDVVLNSLSGEFVDSSLDLLAHGGRFIEMSTDIRDA GRIAAERPGTTYQAFHLVDADPDRLREILTELLALFDQGILDPLPVQAWDIRQAREAFSWMSRARHTGKL VLTIPQHIDPDGTVLITGGSGGLAGVVARHLVADKGARRLLLLSCDTLDATLAAELTESGARVDTAVCDVS DRAALAQVLAGVSPEHPLTAIVHAGGAAVADESRQLHHLTKNRDLAAFVVFSQDAPAATEAFAGIRQAEG LPVTTIAWGIPEAEPVVVGQHLLDRAMASADRAHVAARVNTAGLRALAAANALPPVLKNLVGAETDGTGH QDWSRRFMVAEAARQQELLDLIRTTVMEILSLPTTARYFPDRTFRENGIDSLTAVELVNSLAKTTGLRLSA TMVFDYPTPTALAGRMREL
In some embodiments of any of the foregoing polyketide synthases, the β-ketone processing domain is a ketoreductase, wherein the ketoreductase (a) includes an amino acid other than tyrosine at the position corresponding to the tyrosine in the conserved YAAAN catalytic motif and does not include the conserved aFG helix in SEQ ID NO:1 ; (b) includes a glutamic acid residue at the position
corresponding to alanine 6632 of S9-pksA ORF (the change in S9) in SEQ ID NO: 2; or (c) does not include the amino acids corresponding to amino acids 3386 to 351 6 of WT S12-pksB ORF of SEQ ID NO: 3.
In some embodiments of any of the foregoing polyketide synthases, the β-ketone processing domain is a dehydratase, wherein the dehydratase includes (a) an aspartic acid at the position corresponding to the glycine at position 4288 in pksB of S679-pksB ORF in the conserved
HXXXGXXXXP motif of SEQ ID NO: 4; (b) a substitution in the conserved LPFXW motif at the position corresponding to position 3066 to 3070 in S12-pksB ORF in SEQ ID NO: 5; (c) a deletion between Pro 6844 and Trp 6874 of S679-pksA ORF of SEQ ID NO: 6; or (d) a substitution or deletion at the positions corresponding to A, B, C, and D of SEQ ID NO: 7.
In some embodiments of any of the foregoing polyketide synthases, the β-ketone processing domain is an enoylreductase, wherein the enoylreductase does not include a lysine at the position corresponding to position 1546 of S12-pksB ORF in SEQ ID NO: 8 and/or the aspartic acid at the position corresponding to position 1568 of S12-pksB in SEQ ID NO: 8 or 9.
In another aspect, the disclosure provides a chimeric polyketide synthase, wherein at least one domain of the polyketide synthase has been modified as compared to a polyketide synthase having the sequence of SEQ ID NO: 10 or 1 1 , wherein the modification results in altered enzymatic activity.
SEQ ID NO: 10
MSREEFIQPIHDLLRVNAERLGDKIAYADSRRELTHAELRTRTGRIAGHLVDLAVERGDRVAILLGNRVETI ESYLAIARAGAIAVPLNPDATGAEVAHFLADSGAVLVITDSAHLDDVRRAAAAVTVVLVDEGPLPAGTRSF AELATAEPPTPARDDLGLDEAAWMLYTSGTTGTPKGVVSTQGSGLWSAANCDVPAWELTENDVLLWPA PLFHSLAHHLCLLATTAVGATARIMSGFVAGEVLHELEEHACTVLVGVPTMYHYLLGAVGEAGPRLPSLK MGLVAGAVSPPALIEGFERVFGVPLLDTYGCTETTGSLTVNRLSGPRMPGSCGQAVPGISLRFVDPHTG AEVAEGEEGELWASGPSLMIGYHGRPDATREVLSDGWYRTGDLARRSETGHVTITGRVKELIIRGGENIH PRDIEAVALELPGVRDAAAAGKQHPVLGEIPALYLVPDADGVDAEAVLAACREKLSYFKVPEEIYRVDAIP RTLSGKVKRAALTEAPAELLSAASGNGSLYRLEWVPAETPPAGTGGPVAVHVTRRAVATGPADLPDQEQ AATWDALRGEQTGPGGPVLIDLDGADIDDARLSALASLGEPQIVVRDDTPLVARLAREKSPALTIPGERA WVLEPDHSGVLQELALVAADTDVRPLRPGEVRIEVRAAGLNFRDVLVALGTDLGDGVFGAEGAGVVLET GSDVRDLRPGDRVFGLLEGGHGSIAIADRRMLAVIPEGWSFATAASVPEVFVIAYYGLVDLAGLRAGESV LIHAATGGVGMAATQIARHLGAQVYATAGVGKQHILRDAGLGDDRIADSRTTDFREAFRDSTQGRGVDV VLNSLKGDFVDASLDLLADGGRFLELGQTDIRDAGEIAAERPGTTYHSFTRMNAGPDRLREIIAELLALFE QGVLRPSPVHTWDIRHAREAFSWMSGGRHTGKMVLTMPQRIDPGGTVLIAGDSEALARIAARHLGVRHL LLDRGVADAAPDAVVCDVSDHDALERVLADLSPEHPLTAVIHTGGAAVTDEIRRLHDLTESLDLTDFVVFS QDAPAAVEAFARSRRAHGLPVRTIAWGIPEADPVVADEHLLGRALASAEQAQIVARVNTAGLRALTAANA LPTLLRNLIRAEPEETGQSAWPHRFEAAGADREEALLDLIRANVVDILSLPTADRYAPDRTFREMGIDSLTA VGLRNSLAKATGLPLPTTMVFDYPTPAVLTARMRELLAGESPAPARTAARAVAQDEPLAIVGMACRLPGG VSSPDDLWRLVAAGTDAISEFPADRGWDVDNLYDPDPDAPGKTYTVLGGFLDGVAGFDASFFGISPREA LAMDPQQRLMLEVSWEAFEHAGIPPRSVRGSDAGVFMGAFPSGYDAGLEEFGMTGDAVSVLSGRVSYF FGLEGPAITVDTACSSSLVALHQASSALRQGECSLALVGGVTVLATPQTFVEFSRQRGLALDGRSKAFAD AADGAGWAEGVGVLVVERLSDARAKGHQIWGVIRGSAVNQDGASNGLSAPNGPSQQRVIRQALANAGL APHEVDVVEAHGTGTTLGDPIEAQAVIATYGQDREQPLLLGSLKSNVGHTQAAAGVSGVIKMVMALQHD TVPATLHVDAPSRHVDWTAGAVELVTENRPWPETGRVRRAGVSSFGISGTNAHVILESAPEQPVSPPEA VAPVVASDRVPLVISAKTPAALAEMENRLRAYLAAAPGADPRAVASTLATARSVFEHRAVLLGENTITGTV AGADPRVVFVFPGQGWQQLGMGRALRESSPVFAARMAECAAALSEFVDWDLFTMLDDPAVIDRIDVLQ PACWAVMMSLAAVWQAAGVRPDAVIGHSQGEIAAACVAGALSLRDAARIVALRSQLLAREMVGHGVMA AVALPADDIPLVDGVWIGACNGPSSTVISGTPEAVEVVVAACEERGARVRRITAAVASHSPLGEKIRTELL GISASIPSRTPVVPWLSTADGIWIEAPLDPAYWWRNLREPVGFGPAVDLLQARGENVFLEMSASPVLLPA MNDAVTVATLRRDDGTPDRMLTALAEAHAHGVIVDWPRVFGSTTRVLDLPTYAFEHQRYWAVSADRPS DAGHPMVETVVPLPASGGVALTGRVSLATHAWLADHAVRGTALLPGTAFVELVTRAATEVDCPVIDELVI EAPLPLTQTGAVQLSTTVGEADESGRRPVTVFSQADGTDAWTRHVTATIGRAASLPDPVAWPPAQAEPV DVTGFYDELAAAGYEYGPAFQGLRAAWSDGDTVYAEVVLAEEQAHEVDRYAVHPALLDAALQAGMVNT AGTGQGVRLPFSWNGIQVHSTGATTLRVAATPLADGWSVRAAADNGRPVATIGSLVTRPVTTDMLGSTT DDLFAVVWTEITAPEPGDPSDVGVFTALPEAGGDPLTQTRALTAQVLQTVQQWLAGEDRPLVVRTGTDL ASAAVSGLVRSAQSEHPGRLILVESDDELTPEQLAGTAGLDEPRIRIDGGHYEVPRLAREDASLTVPEDR AWLLELPGSGTLRDLRVIPTDTAERPLRWGEVRVGVRAGGLNFRDVVVALGMVTDPRPAGGEAAGVVL ETGPGVEDLSPGDRVFGILDGGFGSVAIADRRLLAVIPDGWSFTTAASIPVVFATAYYGLVDLAGLRAGES VLIHAATGGVGMAATQIARHLGAEIYGTAGIAKQHVLRDAGLGDDRIADSRTTGFRETFRDSTQGRGVDV VLNSLSGDFVDASLDVLAEGGRFIEMGKTDIRDAEQITHATYRAFDLMDAGPDRVREIIAELLGLFEQGVL RPLPVQAWDIRQARDAFTWMSRARHIGKIVLTIPQQLDPDGTVLISGGSGVLAGILARHLVAERGVRHLLL VSRSAPSEALISELTALGAQVETVACDVSDRVALEQVLDGVPLTAVFHTAAALDDGVVESLTPQRVDTVL RPKADAAWYLHELTRDADLAAFVMYSSVAGIMGAAGQGNYAAANAFLDALAAHRRREGLPALSLAWGL WEDASGLSAGLTETDHDRIRRGGLEAIAAEHGMRLFDTATRQGEPVLLASPLNLTRQGEVPALLRTLHRP VARRAATANGRPADLTPEALLKLVCGRAAAVLGHVDADAVPVAVAFRDLGVDSLTAVELRNSLAKATGLR LPATLVFDYPTPTVLAGRLGELLAGGTAPVRAAVVRRAAASDEPLAIVGMACRLPGGVLSPEDLWRLVES GGDAISGFPVDRGWDVENLFDPDPDAAGRTYAVRGGFLDGAAGFDASFFGISPREAQAMDPQQRLVLE VSWEAFERAGIEPGSVRGSDTGVFMGAYPGGYGVGTDLGGFGMTSVAVSVLAGRVSYFFGLEGPAMT VDTACSSSLVALHQAGSALRQGECSLALVGGVTVMPTPQTFVEFSRQRGLAADGRCKAFADAADGTGF SEGVGVLLVERLSDAQARGHNILAVVRGSAVNQDGASNGLTAPNGPSQQRVIRQALANAGLAGAEVDVV EAHGTGTTLGDPIEAQAVIATYGQDRDQPVLLGSLKSNLGHTQAAAGVSGVIKMVMALRHDTVPATLHID EPSRHIDWTAGAVELVTENQSWPETGRARRAAVSSFGISGTNAHVILESAPAQPVPLVDTPVSAVTAGVV PLPISARTVPALADLEDRLRAYLTTTPETDLPAVASTLAVTRSVFEHRAVLLGEETVTGIAVSDPRVVFVFS GQGSQRVGMGEELAAAFPLFARLHRQVWDLLDVPDLEVDDTGYVQPALFALQVALFGLLESWGVRPEA VIGHSVGEVAAGYVAGVWSLEDACTLVSARARLMQALPAGGAMVAVPVSEERARAVLVDGVEIAAVNGP ASVVLSGDESAVLRVAEGLGRWTRLSASHAFHSVRMEPMLEEFRQVASELTYREPRIVMAAGEQVTTPE YWVRQVRDTVRFGDQVAAFGDAVFLEIGPDRTLSRLIDGIPTLHGDDEQHAVVAALAELHVQGVPIDWSS ILGANPARVLDLPTYAFQHERYWMVSTGRVGGEGHPLLGWGVPVAEAGGRLYTGRVARQDGPVLSVAA FVEMAFAAAGGRPIRELSVDALLYIPDDGTAELQTWVSEHRLTIHARYRDTEPWTRLATAALDTTAPATTH TPHPGLITTALTLTGDEAPAIWHDLTLHTSNATELHTHITPGDDGTLTITATDTTGQPVLTAHTATPTTIPVH TPTTPADDLLTLTWTQIPTPGPGDPTDIAVCTALPDPDGDPLAQTRTLTAQVLQSIQTTLTGEDRPLVVHT GTGLASAAVSGLVRSAQSEHPDRFILVESDDSLPQAQLAAVAGLDEPWLRITGSCYEVPRLTKTTTATAT AVSEPVWNPDGTVLITGGSGALAGILARHLVTERGVRHLLLISRSTPSTTLTDELRELGAHVDVAACDVSD RDALARVLDGVDLTAVFHTAGALDDGVVESLTPQRLDTVLTPKADGAWHLHELTRDRDLTAFVMYSSAA GVMGAAGQGNYAAANAFLDALAEHRHADGLPALSLAWGMWDDTDGMTASLSGTDHRRIRRSGQRAIT AEHGMRLLDKASGRSEPVLVATAMNPIPDTDLPALLRSLYPKTARKSQPIQELSPEALLKIVRDSAALMLG HPNTDAIAATTAFRDLGVDSLIAVELRNSLAKATGLRLPATLVFDYPTPTVLAGRLGELLAGVTPQRHATV RTGTASDEPLAIVGMACRLPGGVSSPEDLWRLVESGTDAITDFPTDRGWDTDDLFDPDPDTAGKTYTVH GGFLDDVAGFDASFFGISPREAQAMDPQQRLVLEAAWEAFERAGIEPGSVRGSDTGVFMGAYPGGYGI GADLGGFGATAGAGSVLSGRLSYFFGLEGPAMTVDTACSSSLVALHQAGSALRQGECSLALVGGVTVIA NPQIFVEFSRQRGLAADGRCKAFADSADGTGWSEGVGVLLVERLSDAQARGHNILAVVRGSAVNQDGA SNGLTAPNGPSQQRVIRQALANAGLAGAEVDVVEAHGTGTTLGDPIEAQAVIATYGQDRDQSVLLGSLKS NLGHTQAAAGVSGVIKMVMALQNGVVPRTLHADQPSRHIDWTAGAVELVTENQPWPELDRPRRAAVSA FGVSGTNAHVILESAPDQPVPLVDTPVSAVTAGVVPLPISARTVPALADLEDQLRAYLTTAPETDLPAVAS TLATTRSVFEHRAVLLGEDTVTGTAIPDPRIVFVFSGQGSQRAGMGEELAAAFPLFARLHRQVWDLLDVP DLDVDDTGYVQPALFALQVALFGLLESWGVRPRAVIGHSVGEVAAGYVAGVWSLEDACALVSARARLM QALPAGGAMVAVPVSEERARAVLVDGVEIAAVNGPASVVLSGDEAAVLRVAEGLGRWTRLSASHAFHSV RMEPMLEEFRQVVSRLTYREPRIVMAAGEQVTTPEYWVRQVRETVRFGDQVAAFGDAVFLEIGPDRTLS RLIDGIAMLDGDDEVRAAVAALAMMHVQGVGVDWPAILGTTTGRVLDLPTYAFQHERYWMANADEGHP LLGKVEHPLLGSVMALPNSDGVVLTGRISLATHAWLADHVVRGTVLLPGTGFVEMVARAAAEVGCGVIDE LLIEAPLLLPEHGGVHLSVSVGEADGAGRRPVTVFAQADDAEVWVRQVTATISPAGPAVSLPELEVWPPV QAEPVDVSTFYERLARADWQWGPAFQGLRAAWRDGDTIYAEIVLADEEAREADQFLVHPALLDAALQTS VLKTPDDLRLPFSWNQIEFHATGAAILRVAVTPVADRWIVHAADSTGRPVATIGALVSRPVTAETLGSNTD DLFALTWTEIPTPGPGDPADVAVCTALPEPDSDPLTQTRTLTAQVLQSIQTSLTGEDRPLVVHTGTGLASA AVSGLVRSAQSEHPDRFILVECDDETLTPDQLAATAGLDEPWLRITGGHYEVPRLTKTTTAAATTVSEPV WDPDGTVLITGGSGALAGILARHLVTERSVRHLLLISRSTPSTTLINELRELGAHIETAACDVSDRDALARV LDGVDLTAVFHTAGALDDGVVESLTPQRLDTVLMPKADAAWHLHELTRDRDLAAFVMYSSAAGVMGAA GQGNYAAANAFLDALAEHRRADGLPALSLAWGMWDDADGMTASLSGTDHRRIRRSGQRAITAEHGMRL LDKASGRSEPVLVATAMNPAGEGEVPALLRTLHRPVARRAATTNGRPADLTPEALLKVVRDSAAVVLGH ASADTVPAATAFQELGLDSLIAVELRNSLAKATGLRLPATMVFDYPTPAALAGRLGELLAGETTPATAAVV RRATASDEPLAIVGMACRLPGGVSSPEDLWRLVESGFDAITGFPTDRGWDVDNLYDPDPDAPGKSTTLH GGFLDDVAGFDASFFGISPREAVAMDPQQRLAMEVSWEAFERAGIEPGSVRGSDTGVFMGAYPGGYGI GAELGGFMLTGRAGSVLAGRVSYFFGLEGPAMTVDTACSSSLVALHQAAYALRQGECSLALVGGVTVM PTPVMFVEFSQQQNLADDGRCKAFADSADGTGWSEGVGVLLVERLSDAQARGHNILAVVRGSAVNQD GASNGLTAPNGPSQQRVIRSALTSAGLTTADVDVVEAHGTGTTLGDPIEAQAVLATYGQDRDQPVLLGSL KSNLGHTQAAAGVSGVIKMVMALQNGVVPRTLHVEEPSRHVDWTAGAVELVTENQSWPETGRARRAAV SSFGFSGTNAHVILESAPAQPVPPMDTPAPAVTTGVVPLPISAKSLPALADLEDQLRAYLTATPETDLPAV ASTLAMTRSVFEHRAVLLGEETVTGTAIPDPRIVFVFSGQGSQRVGMGEELAAAFPLFARLHRQVWDLLD VPDLDVDDTGYVQPALFALQVALFGLLESWGVRPRAVIGHSVGEVAAGYVAGVWSLEDACALVSARARL MQALPAGGAMVAVPVSEERARVALVDGVEIAAVNGPASVVLSGDEAAVLQIAEGLGRWTRLSASHAFHS VRMEPMLEEFGQVASELTYQEPRIVMAAGEQVTTPEYWVRQVRDTVRFGDQVAAFGDAVFLEIGPDRTL SRLIDGIAMLDGDDEVRAAVAALAELHVQGVPIDWPAVLGTTTGRVLDLPTYAFQHQRYWAASTDRPAG DGHPLLDTVVALPGADGVVLTGRISLATHAWLADHAVRGTVLLPGTGFVEMVARAAAEVGCAVVDELVIE APLLLPASGGVQLSVSVGEADDAGHRPVTVHSQADETEAWVRHVTATISPSGPIVSPPEFEVWPPAQAE PVEVARFYDELAAAGYEYGAAFQGLRAAWRAGETIYAEVVLAEDQTLEAARFTVHPALLDAALQANILNA SGDLRLPFSWGQVQFHTTGAATLRVAVTPVADGWTIQATDDAGRPVATVGSVVARPVAGLGATAEDLFA LTWNEIPAPGQGGRTVGRFEDLADDGPVPELVVFTALPDVDADPLVRTRALTARVLEAIQRWLGEPRFA DSTLVVRTGTDLASAAVSGLVRSAQSEHPDRFILVEGDSSPVEIGLDEPWLRVDGGRYEVPRLIRLSAEP VQEAAWNPDGMVLITGGTGALAGILARHLVAENKARRLLLVSRSVPDDALISELTELGAEVGTAVCDVSD RAALARVLAGVPSLTAVIHTAGVLDDGVMESLTPQRLDTVLRAKADGAWHLHELTRDRDLAAFVMYSSA AGLMGSPGQGNYAAANAFLDALAVERRAEGLPALSLAWGFWEETTGLTANLTGADRDRIRRGGLQTITA ERGMRMFDTATQHGEPVLLAAPISPVRDGEVPALLRSLHRRGTRRGTTADASAQWLAGLAPEEREGALI KVVRDTAAVVLGHADAGTIPVTAAFKDLGLDSLTAVELRNSLAKSTGLRLPATMVFDYPTPASLAARLDDL MNPRVSSTALLAELDRIEGMFDSVTFDEKQASLVKDRLSAALGKWQQISRSADVATVALANADAGEILDFI DREFGNPTI SEQ ID N0: 1 1
MPDHDKLVEYLRWATAELHTTRAKLQAATEAGTQPLAIVGMACRLPGGVSSPEDLWRLVESGTDAISGF PVDRGWDVDGLYDPDPDVPGKSYTVEGGFLDAVTGFDAPFFGISPREALAMDPQQRLVLEASWEAFER AGIEPGSVRGSDTGVFMGAFPGGYGTGADLGGFGMTGGAASVLSGRVSYFFGLEGPAMTVDTVCSSSL VALHQAGYALRHGECSLALVGGVTVMSTPQTFVEFSRQRGLAADGRCKAFADNADGTGWSEGVGVLLV ERLSDAQARGHNILAVVRGSAVNQDGASNGLTAPNGPSQQRVIRQALANAGLTGADVDVVEAHGTGTTL GDPIEAQAVIATYGRDRDQPVLLGSLKSNLGHTQAAAGVSGVIKMVMALQNGVVPRTLHIEEPSRHVDWT AGAVQLVTENRPWPELGRARRAAVSSFGLSGTNAHVILESAPDQPPAPTTDTPVSAVTAGVVPLPISAKT VPALADLEDRLRTYLTTTPDTDLPAVASTLATTRSLFEHRAVLLGEDTVTGTAIPDPRVVFVFPGQGWQW QGMGSALLTSSTVFAERMAECAAALSEFVDWDLLTVLDDPSVVDRVDVVQPACWAVMISLAAVWQAAGI HPDIVLGHSQGEIAAACLAGAISLPDAARIVAQRSQLIAHQLTGHGAMASISLPADDIPTTDKVWIAAHNGT STVIAGDPQAVEAVLATCETRGARVRKINVDYASHTPHVEQIRTELLDITTGIEAHTPAVPWLSTTDNTWID QPLDPTYWYRNLREPVRFGPAIDLLQTQDNNLFIEISASPVLLQTMDNAATVATLRRDEDTTQRLLTAFAE AHVHGATIDWPTVLDTTTTPVLDLPTYPFQRQRYWATSNGRSTGQGHPLLETVVALPGTDGVALTGRISL ATHPWLTDHTVRGTVLLPGTAFVELVTRAATEVNCQIIDELIIEAPLPLPQTDGVQLSVTVGEADEAGHRP VTVYSQTDESDDWIQHVTATIGPGASLPETAAWPPAHAEPVNVTGLYDNLAAAGYEYGPAFQGLQAAW RAGDTVYAEVTLAEEQAQETARFTMHPALLDAALHTIALHDTGDLHLPFSWTRVQFHGTGAATLRVAVTP AADGWNIRATDDTGRAVATIGSLVTRPMAAETTDDLLALTWTEIPAPEPVDPTDVVVFTALPDTVEDVPA QTRALTTRVLHTIQEWLADDDRTLIVRTGTDLASAAVSGLVRSAQSEHPGRFILVESADEALTQEQLAATA GLDEPRLRITGGRYEVPRLTREDTALAVPTDRAWLLEQPRSGSLEDLALLPTDAAERPLQAGEVRIGVRA AGMNFRDVVVALGMVTDTRLAGGEAAGVVLEVGTDVNDFRPGDRVFGILEGGFGSVAICDHRTLAVIPD GWSFTTAASVPIAFATAYYGLVDLAGLRAGESVLIHAATGGVGIAATQIARHLGAEIYGTASVGKQHVLRD AGLADDRIADSRTTDFRDTFRDGTQGRGVDVVLNSLRGEFIDASLDLLVDGGRFIEMGKTDIRDAAQIPDA TYHAFDLMDAGHDRLREIMTELLALFEQGVLHPMPVHAFDIRQAREAFSWMSRARHIGKLVLTIPQPIDPD GTVLITGGSGVLAGIVARYLVTENRARHLLLLSRSAPSASLIDELTALGAHVDVAACDVADRAALAEILDGV DLTAVIHTAGALDDGVVESLTPQRLDTVLTPKADGAWHLHELTRDRDLAAFIVYSSAAGVLGAAGQGNYA AANAFLDALAVHRRLEGLPGLSLAWGLWEDASGLTADLTDADRDRIRRSGQRAITAAYGMRMLDAATRQ SEAILLAAPISPIQDGDVPAILRSLHRRVGRRASVAHGHPADLTPEALLKVVRDSAAMVLGHTNADTVPTA TAFQELGLDSLTAVELRNSLTKATGLRLPATMAFDYPTPDALAARLGELLAGEAAPKAAAAVRRATASDE PLAIVGMACRLPGGVSSPEDLWRLVESGTDAITDFPTDRGWDTDTLFDPDPDTPGKTYTVHGGFLNDVA GFDAPFFGISPREAVAMDPQQRLVLESSWEAFERAGIQPDSIRGSDTGVFMGAYPDGYGIGADLAGFGV TAGAGSVLSGRVSYFFGLEGPAMTVDTACSSSLVALHQAAYALRQGECSLALVGGVTVMPSPRTFIEFS RQRGLAADGRSKAFADAADGTGFSEGVGVLLVERLSDAQAKGHNILALVRSSAVNQDGASNGLTAPNG PSQQRVIQSALAGAGLTSADVDVVEAHGTGTTLGDPIEAQAVLATYGQDRDQPVLLGSLKSNLGHTQAA AGVSGVIKMVMALQHNTVPATLHVDAPSRHVDWTAGAVRLATENQPWPETNRPRRAGVSSFGVSGTNA HVILEQAPAASPVEPVDTTDVVIPLVVSARSSGSLSDQADRLAALVGSPDAPALTSLADALLTRRTVFSQR AVVVAGSHEQAAAGLRALASGDSHPALVTGAAGPARGVVLVFPGQGSQWAGMGAELLDTSPVFAARIA ECAEALRPWVDWSLDEVLRGDASADVLGRVDVVQPASFAVMVGLAAVWESAGVRPDAVLGHSQGEIAA AYVAGALSLTDAAKIVAVRSRLIAARLAGRGGMASVALAPDEAAAKLGRTELAAVNGPASVVIAGDAEALD ETLAMLEGEAVRVRRVAVDYASHTPHVEELEQSMAEALADVRSRQPRVGFLSTVTGDWVTEAGALDGG YWYRNLRQPVRFGPAVASLAEAGYTVFVEASAHPVLVQPVAETLDRTDAVVTGTLRRQDGGLPRLLTSM AELFVGGVPVNWPVLLPAGAVRGWVDLPTYAFDHQRYWLENRVATDAAALGLAGADHPLLGAIVAVPQ SGGVAMTSRLSPRNHPWLAEHTLGGVPTVPTSVLVELAVRAGDEVGCGVVEELTVDAPLLLPERGGVRV QVIVGATDANGQRGLDIFSAPEDTGQEAWTRHATGTLAPGGDIAADVDLSAWPPANAQPVDVTDGYDLL ERAGYGYGPAFQGVRAIWRRGEELFAEVALEPELTDTAARFGLHPALLDAAWHPELRDEVAETSPDGRR WWSQPSRWAGLRLHTAGATVLRVRLAPVDADSMSLQAADETGDPVLTVDSLSLCAVSADQLTTAESSD DALFRLEWTPLSKAPTAARSWVPVETGADVAALDGQAVVDAVMLEAAGTGDALELTCRVLEVVQAWLTL PGWDESRLVVVTRGAVGAVGDPAGSAVWGLVRAAQAENPDRIALLDLDGGRPVEPLLAESEPQLAIRGA EALVPRLIRAAAATDAPALFDESQTVLITGGTGSLGGLLARHLVGRYGLRRLVLVSRRGPDAPGAYELAAE LAAHGAEAALVACDLTDRDAVARLLTEHHPTAVVHAAGVSDDGVIGTLTSDRLAYVFGPKATAARHLDEL TRELLPDLAAFVTYSSISAVFLGAGSGGYAAANAYLDGLMARRHAEGLPGLSLAWGLWDQEADGGGMA AGLQDITRNRMRRRGGVLSFTPAEGMALFDAAMATDEALVVPVRLDLPALRAEAVAEGRSAPVLLRGLV RPGRRLARTVSGGTGVLADLTPEALLKLVRGRAAAVLGHVDADAVPVAAAFKDLGVDSLTAVELRNSLAK ATGLRLPATLVFDYPTPTVLAGRLGELLAGGTAPVRAAVVRRAAASDEPLAIVGMACRLPGGVLSPEDLW RLVESGGDAISGFPVDRGWDVENLFDPDPDAAGRTYAVRGGFLDGAAGFDASFFGISPREAQAMDPQQ RLVLEVSWEAFERAGIEPGSVRGSDTGVFMGAYPGGYGMGTDLGGFGMTSVAVSVLAGRVSYFFGLEG PAMTVDTACSSSLVALHQAGSALRQGECSLALVGGVTVMPTPQTFVEFSRQRGLAADGRCKAFADAAD GTGFSEGVGVLLVERLSDAQARGHNILAVVRGSAVNQDGASNGLTAPNGPAQQRVIQSALAGAGLASAD VDVVEAHGTGTTLGDPIEAQAVIATYGQDRDQPVLLGSLKSNLGHTQAAAGVSGVIKMVMALQNGVVPR TLHIDEPSRHIDWTAGAVELVTENQSWPETGRARRAAVSSFGISGTNAHVILESAPAQPVPLVDTPVSDV TAGVVPLPISARTVPALADLEDQLRAYLTTAPETDLPAVASTLAMTRSVFEHRAVLLGEETVTGIAVSDPR VVFVFSGQGSQRVGMGEELAAAFPLFARLHRQVWDLLDVPDLEVDDTGYVQPALFALQVALFGLLESW GVRPRAVIGHSVGEVAAGYVAGVWSLEDACTLVSARARLMQALPAGGAMVAVPVSEERARAVLVDGVEI AAVNGPASVVLSGDESAVLRVAEGLGRWTRLSASHAFHSVRMEPMLEEFRQVASELTYREPRIVMAAGE QVTTPEYWVRQVRDTVRFGDQVAAFGDAVFLEIGPDRTLSRLIDGIAMLDGDDEVRAAVAALAMMHVQG VGVDWPAVLGTTTGRVLDLPTYAFQHERYWMVSTGRPGGEGHPLLGWGVPVAEADGRLYTGRVARQD GPVLPVAAFVEMAFAAAGGRPIRELSVDALLYIPDDGTAELQTWVSEHRLTIHARYRDTEPWTRLATATLD TTEPATTHTPHPGLITTALTLTGDEAPAIWHDLTLHTSNATELHTHITPGDDGTLTITATDATGQPVLTAHAA TPTTIPVHTPTTPADDLLTLTWTQIPTPGPGDGADIAVCTALPDPDSDPLAQTRTLTAQVLHSIQASLTGED RPLVVHTGTGLASAAVSGLVRSAQSEHPDRFILVESDETLTPDQLAAVAGLDEPWLRITDGRYEVPRLTK TTTTATATAVSEPVWDPDGTVLITGGSGALAGILARHLVTERGVRHLLLVSRSTPSTTLIDELRELGAHVDV AACDVSDRAALARVLDGVDLTAVFHTAGALDDGVVESLTPQRVDAVLRPKADGAWHLHELTRDRDLTAF VMYSSAAGVMGAAGQGNYAAANAFLDALAEHRRADGLPALSLAWGMWDDADGMTASLSGTDHRRIRR SGQRAITAEHGMRLLDKASGRSEPVLVATAMNPIPDTDLPALLRSLYPKTARKSQPIQELSPEALLKIVRDS AAMVLGHANADTVPTATALQELGLDSLTAVELRNSLTKATGLRLPATMAFDYPTPAALAGRLGELLAGDT TPATAAVVRRATASDEPLAIVGMACRLPGGVSTPEDLWRLVESGTDAITDFPTDRGWDTDDLFDPDPDT PGKTYTVHGGFLDDVAGFDASFFGISPREALAMDSQQRLVLEAAWEAFERAGIEPGSVRGSDTGVFMGA YPDGYGIGADLGGFGATAGAGSVLSGRLSYFFGLEGPAMTVDTACSSSLVALHQAGSALRQGECSLALV GGVTVIANPQIFVEFSRQRGLAADGRCKAFADNADGTGFSEGVGVLLVERLSDAQAKGHNILALVRSSAV NQDGASNGLTAPNGPSQQRVIRQALANAGLTGAEVDVVEAHGTGTTLGDPIEAQAVLATYGQDRDQPVL LGSLKSNLGHTQAAAGVSGVIKMVMALRHDTVPATLHIDEPSRHIDWTAGAVELVTENQPWPVLGRPRR AAVSAFGVSGTNAHVILESAPDQPPAPATDTPAPAATAGVVPLPISAKTVPALADLEDRLRTYLTTTPETDL PAVASTLATTRSLFEHRAVLLGEDTVTGTTIPDPRIVFVFPGQGWQWQGMGSALLTSSTVFAERMAECA AALSEFVDWDLLTVLDDPSIVDRVDVVQPACWAVMISLAAVWQAAGIHPDIVLGHSQGEIAAACLAGAISL PDAARIVAQRSQLIAHQLTGHGAMASISLPADDIPTTDKVWIAAHNGTSTVIAGDPQALDTVLATCETHGA RVRKINVDYASHTPHVEQIRTELLDITTDIEAHTPTVPWLSTTDNTWIDQPLDPTYWYRNLREPVRFGPAID LLQTQDNNLFIEISASPVLLQTMDNATTVATLRRDEDTTQRLLTAFAEAHVHGATIDWPTVLDTTTTPVLDL PTYPFQRQRYWATSNGRPTSQGHPLLETVVALPGTHGVALTGRISLATHPWLTDHTVRGTVLLPGTAFV ELVTHAATEVNCQVIDELIIEAPLPLPQNGGVQLSVTVGEADEAGHRPVTVYSQTDESDDWVQHVTATIAP GVSSSESAAWPPAQAEPVNVTGLYDNLAAAGYEYG PAFQGLQTAWRDGSTVYAEVTLAEEQAQETARF TMHPALLDAALHTIALHDTADLQLPFSWRQVQFHGSGAATLRVAVTPAADGWNIRATDDTGQTVATIGSL VTRPMAAETTNDLLALTWTEIPAPEPVDPADVVVFTALPEPGSDPLAQTRALTTRVLHTIQEWLADDDRTL IVRTGTDLASAAVSGLVRSAQSEHPGRFILVESDDETLTHEQLAATAGLDEPRLRITDGRYEVPRLTREDT ALAVPEGGAWMLDQPSRSGTLQDLRLVPTDAAERPLRPGEVRVGVRAAGLNFRDVAVALGMVTDTRLI GGEGAGVVLEAGPGVEDLRPGDRVFGLLEGGFGPVAVADRRALALIPDGWSFTTAASVPIAFATAYYGLL DLAGLRAGESVLIHAATGGVGMAATQIARHLGADVYATASTGKQHVLRDAGLSDDRIADSRTTGFRETFR DSTDGRGVDVVLNSLKGDFVDASLDLLVDGGRFIEMGKTDIRDAAQIPDATYRAFDLMDAGPERLREIITE LLALFEQGVLRPLPVHAFDIRQARDAFGWMSRARHIGKLVLTIPQPIDPDGTVLITGGSGVLAGIVARHLVI AEGLRNLLLLSRSAPSEALIGELTALGAQVETAACDIADRAALARVLDGVPLTAVIHTAGALDDGVVESLDP QRLDSVLTPKADGAWHLHELTRDRDLAAFIMYSSAAGVLGAAGQGNYAAANAFVDALAVHRRFMGLPAL SLAWGLWDDTSALTAGLTDSDHDRIRRSGARTITAEHGMRMFDAATRQSEAVLLAAPMGPIRGEDVPAL LRGLATVRQPRTRAKRDMGPERLRDRLNGRTSVEQHRIMVELVLAHATSVLGHESPDAIAPDRAFKDLG MDSLTAIELRNHLVAETGVRLPATTAFDHPTADDLAKRLLAEVGLTPAPQRTEADIREEVVVREPAGDDS WTSEPIAIVSMSCRAPGGVDSPESLWRLVESGTDAITDFPGDRGWDVAGLYSPDPDTGYKTYCVQGGFL DAAADFDAAFFGISPREALGMDPQQRLLLETSWEAIERARIDPRSLRGRNVGVYVGGAAQGYGVGAIDQ QRDNVITGSSISLLSGRLSYALGLEGPGVTVDTACSSSLVALHLACQALRQRECSMALVSGVSVIPTPDVF VEFSRQRGLAADGRCKSFSASADGTIWAEGVGVLVLERLSEATRLGHRVLAVVRGSAVNSDGASNGLTA PNGVSQQRVIRQALTGAGLTAADVDVVEAHGTGTKLGDPIEAEAILATYGQDRSTPVCLGSLKSNIGHAM AASGVLAVIKMVEAMRHGLIPRTLHVEEPSPHVDWASGDVALLTENQPWPDDAKLRRAGVSSFGLSGTN AHVVLEQYRAPAAPDITTTEHEPLAWTLSARDPKALREQAGRLHAALTESPQWRPLDIGYSLATTRSNFA HRAVAVGSDREDLLRALSKLADGSAWPALVTATAKDRRVAYLFDGQGSQRPDMGSGLYERFPAFARAW DRISAEFGKHLDHSLTDVYLGRGDAATADLVDDTLYAQAGLFTMEIALFELLAEWGVRPDFVSGHSIGETA AAYAAGVLSLEDVTTLIVARGRALRQVPPGAMVALRAGEDEAREFLGRTGAALDLAAVNSPTSVVVSGAS EAVAGFRARWTESGREARTLNVRHAFHSRHVEAVLGEFREVLESLTFRTPALPVVSTVTGRLIEPTELST SEYWLRQVRQTVRFHDAVRELSGQGVGTFVEIGPSGALASAGLECLGDEASFHAVQRPGSPGDVCLMT AVAELHAGGTTVDWATVLAGGRATDLPVYPFQHGSYWLAPVTRAADGAPSAGVPAPGEYARPSAPEEP RTMLELVRLEAAIALSITDPGLIADDSSFLDLGFDSISALRLSNRLAAVTGLDLPPSLLFDHPTPAELAARLD ELSAADLDGAGVYALLEEIDELDDEDLDMTEEEQTAISELLTKLSAKWSR
In another aspect, the disclosure provides, a chimeric polyketide synthase, wherein at least one ketoreductase domain (a) includes an amino acid other than tyrosine at the position corresponding to the tyrosine in the conserved YAAAN catalytic motif and does not include the conserved aFG helix in SEQ ID NO: 1 ; (b) includes a glutamic acid residue at the position corresponding to alanine 6632 of S9-pksA ORF in SEQ ID NO: 2; or (c) does not include the amino acids corresponding to amino acids 3386 to 3516 of WT S12-pksB ORF of SEQ ID NO: 3.
In another aspect, the disclosure provides a chimeric polyketide synthase, wherein at least one dehydratase domain (a) an aspartic acid at the position corresponding to the glycine at position 4288 in pksB of S679-pksB ORF in the conserved HXXXGXXXXP motif of SEQ ID NO: 4; (b) includes a substitution in the conserved LPFXW motif at the position corresponding to position 3066 to 3070 in S12- pksB ORF in SEQ ID NO: 5; (c) includes a deletion corresponding to positions between Pro 6844 and Trp 6874 of S679-pksA ORF of SEQ ID NO: 6; or (d) includes a substitution or deletion at the positions corresponding to A, B, C, and D of SEQ ID NO: 7.
In another aspect, the disclosure provides a chimeric polyketide synthase, wherein at least one enoylreductase domain does not include a lysine at the position corresponding to position 1546 of S12- pksB ORF in SEQ ID NO: 8 and/or the aspartic acid at the position corresponding to position 1 568 of S12-pksB in SEQ ID NO: 8 or 9. In another aspect, the disclosure provides a chimeric polyketide synthase including a domain having at least 80% sequence identity to the amino acid sequence of (a) SEQ ID NO: 12, 13, or 14; (b) SEQ ID NO: 15, 1 6, or 17; (c) SEQ ID NO: 18, 19, or 20; (d) SEQ ID NO: 21 , 22, or 23; (e) SEQ ID NO: 24, 25, 26, or 27; (f) SEQ ID NO: 28, 29, 30, or 31 ; (g) SEQ ID NO: 32, 33, 34, or 35; or (h) SEQ ID NO: 36 or 37.
SEQ ID NO: 12:
CGCGACCGGGACTTGGCCGCGTTCGTCATGTACTCCTCCGCGGCCGGTGTGATGGGTGCTGAGGG CCAGGGCAACTACGCGGCGGCCAACGCGTTCCTCGATGCCCTGGCCGAGCACCGCCGC
SEQ ID NO: 13:
TCATGTACTCCTCCGCGGCCGGTGTGATGGGTGCTGCGGGCCAGGGCAACTTCGCGGCGGCCAAC GCGTTCCTCGATGCCCTGGCCGAGCACCGCCGCGCTGACGGCTTGCCCGCACTCTCCCTGGCATG GGGTATGTGGGACGACGCAGACGGTATGAGCGGTCAGCGGGCCATCACCGCCGAACACGGGAT
SEQ ID NO: 14:
GGCGTCGACCTGACCGCGGTGTTCCACACCGCCGGAGCCCTGGACGACGGTGTCGTGGAACTGGT CGCCACCGCAATGAACCCGGCGGGGGAGGGTGAAGTCCCCGCGCTGCTGCGTACG SEQ ID NO: 15:
CGCGACCGGGATCTGGCGGCGTTCGTCATGTACTCCTCCGCCGCGGGCCTCATGGGCAGCGAGGG ACAGGGCAACTACGCGGCAGCCAACGCCTTCCTGGACGCGCTCGCGGTAGAGCGTCGT
SEQ ID NO: 16:
TCATGTACTCCTCCGCCGCGGGCCTCATGGGCAGCCCCGGACAGGGCAACTTCGCGGCAGCCAAC GCCTTCCTGGACGCGCTCGCGGTAGAGCGTCGTGCGGAGGGTTTGCCCGCGCTCTCGCTGGCGTG GGGTTTCTGGGAGGAAACGACCGGCCTGGGGGGATTGCAGACCATCACCGCCGAGCGCGGCAT
SEQ ID NO: 17:
GTGCCGTCCCTGACGGCGGTGATCCACACCGCGGGAGTCCTCGACGACGGGGTGATGGAATTGCT TGCCGCACCGATGGCCCCGGTCCGGGACGGCGAGGTTCCCGCCCTGCTGCGGTCG
SEQ ID NO: 18:
GCGGCGGTGTACGGCCAGAGCGTCCAGGAACGCGTTGGCCGCAGCGTAGTTACCTTGTCCCTCAG CGCCCAGGACGCCGGCGGCGGACGAGTACACGATGAATGCGGCCAAGTCCCTGTCGCG
SEQ ID NO: 19:
ATCCCGTACGCGGCGCTGATGGCACGCTGGCCGCTCAGGCCGCTCGCGTCCTCCCACAGTCCCCA GGCCAGGGACAAACCAGGCAAACCCTCAAGGCGGCGGTGTACGGCCAGAGCGTCCAGGAACGCGT TGGCCGCAGCGAAGTTACCTTGTCCGGCAGCGCCCAGGACGCCGGCGGCGGACGAGTACACGA SEQ ID NO: 20:
TGACCGCAGGATCGCGGGGACGTCCCCGTCCTGGATCGGGCTGATCGGCGCGGCGAGCAGTTCCA CGACACCGTCGTCGAGGGCGCCGGCGGTGTGGATCACCGCGGTCAGGTCGACGCC SEQ ID NO: 21 :
GTGCCGCCTGGCCATCAGGCCGTCGAGGTAGGCGTTCGCGGCCGCGTAACCGCCGGAGCCCTCG CCCAGGAACACCGCGGAGATGGAGGAGTAGGTGACGAACGCCGCCAGGTCGGGGAGCAA
SEQ ID NO: 22:
ATGCCCTCGGCCGGGGTGAACGACAGCACGCCGCCCATGCCGCCACCGTCGGCTTCCTGGTCCCA CAGGCCCCACGCCAGGGACAGGCCGGGCAGCCCTTCGGCGTGCCGCCTGGCCATCAGGCCGTCG AGGTAGGCGTTCGCGGCCGCGAAACCGCCGGAGCCCGCGCCCAGGAACACCGCGGAGATGGAGG
SEQ ID NO: 23:
GAGCACCACCCGACCGCGGTCGTGCATGCGGCTGGCGTGTCCGACGACGGCGTGATCGGCGTGG TGCCGGTCCGGCTCGACCTGCCCGCCCTCCGCGCCGAAGCGGTCGCCGAGGGCCGC
SEQ ID NO: 24:
GAGGCGCGGGAAGCAGACCAGTTCCTGGTGCACCCCGCCCTGCTGGACGCGGCCTGGCATCCGG AGCTGCGCGACGAAGTGGCCGAGACGAGCCCGGACGGCCGGCGCTGGTGGTCGCAACCGTCGCG ATGGAACCAGATCGAGTTCCACGCGACCGGCGCGGCGATACTGCGCGTC
SEQ ID NO: 25:
GAGGCGCGGGAAGCAGACCAGTTCCTGGTGCACCCCGCCCTGCTGACCACCGCCCTCACCCTCAC CGGCGACGAGGCACCCGCCATCTGGAACCAGATCGAGTTCCACGCGACCGGCGCGGCGATACTGC GCGTC
SEQ ID NO: 26:
GTCACGGCCACGATCAGCCCCGCCGGCCCTGCCGTCTCGCTGCCGGCCTTCGCGGGTGGCGAACC CCTGCACATCGCGGACGGCACCCCGGCCGGCTTCCTCCTGCATCCGGACGCGACACCGGCCGCCG ACTGGAACCAGATCGAGTTCCACGCGACCGGCGCGGCGATACTGCGC
SEQ ID NO: 27:
CTCGGTTCGGTGATGGCGTTGCCGAACTCGGACGGTGTGGTGCTGACCGGCAGGATCTCGCGTCA GGACGGTCCGGTTCTGTCCGTTGCGGCTTTCGTTGAAATGGCGTTCGCGGCTGCTGGTGGTCGCCC GATCCGTGAACTGTCTGTTGACGCGCTGCTGTACATCCCGGACGACGGCACCGCGGAACTGCAGAC CTGGGTCTCTGAACACCGTCTGACCATCCACGCACGTTACCGTGACACCGAACCGTGGACCCGTCT GGCGACCGCCGCTCTGGACACCACCGCGCCTGCGACGACCCACACCCCGCACCCTGGTCTGATCA CCACGGCGCTGACCCTGACCGGTGACGAAGCACCGGCGATCTGGAACCAGATCGAGTTCCACGCG ACCGGCGCGGCGATACTGCGCGTCGCGGTGACACCGGTG SEQ ID NO: 28:
CAGACGCTGGAGGCGGCCCGGTTTACGGTGCATCCCGCGCTGCTGGACGCGGCCTGGCATCCGGA
GCTGCGCGACGAAGTGGCCGAGACGAGCCCGGACGGCCGGCGCTGGTGGTCGCAACCGTCGCGA
TGGGGTCAGGTTCAGTTCCATACGACCGGCGCGGCGACGCTGCGGGTC
SEQ ID NO: 29:
CAGACGCTGGAGGCGGCCCGGTTTACGGTGCATCCCGCGCTGCTGACCACCGCCCTCACCCTCAC
CGGCGACGAGGCACCCGCCATCTGGGGTCAGGTTCAGTTCCATACGACCGGCGCGGCGACGCTGC
GGGTC
SEQ ID NO: 30
GTCACGGCCACGATCAGCCCGTCCGGTCCGATCGTCTCGCCGCCGGCCTTCGCGGGTGGCGAACC CCTGCACATCGCGGACGGCACCCCGGCCGGCTTCCTCCTGCATCCGGACGCGACACCGGCCGCCG ACTGGGGTCAGGTTCAGTTCCATACGACCGGCGCGGCGACGCTGCGG
SEQ ID NO: 31 :
CTGGACACCGTCGTGGCGTTGCCGGGCGCGGACGGTGTGGTGCTGACCGGCAGGATCTCGCGTCA GGACGGTCCGGTTCTGTCCGTTGCGGCTTTCGTTGAAATGGCGTTCGCGGCTGCTGGTGGTCGCCC GATCCGTGAACTGTCTGTTGACGCGCTGCTGTACATCCCGGACGACGGCACCGCGGAACTGCAGAC CTGGGTCTCTGAACACCGTCTGACCATCCACGCACGTTACCGTGACACCGAACCGTGGACCCGTCT GGCGACCGCCGCTCTGGACACCACCGCGCCTGCGACGACCCACACCCCGCACCCTGGTCTGATCA CCACGGCGCTGACCCTGACCGGTGACGAAGCACCGGCGATCTGGGGTCAGGTTCAGTTCCATACG ACCGGCGCGGCGACGCTGCGGGTCGCGGTGACGCCGGTG SEQ ID NO: 32:
CGACCCGTAGCGTCGCCGCGCCGGTACCGTGGAACTGCACCCGAGCCATCGCGACGGTTGCGACC
ACCAGCGCCGGCCGTCCGGGCTCGTCTCGGCCACTTCGTCGCGCAGCTCCGGATGCCAGGCCGC
GTCGAGCAGGGCGGGATGCATGGTGAAGCGGGCCGTTTCCTGGGCCTG SEQ ID NO: 33:
CGACCCGTAGCGTCGCCGCGCCGGTACCGTGGAACTGCACCCGAGCCAGATGGCGGGTGCCTCGT
CGCCGGTGAGGGTGAGGGCGGTGGTGAGCAGGGCGGGATGCATGGTGAAGCGGGCCGTTTCCTG
GGCCTG SEQ ID NO: 34:
CCGTAGCGTCGCCGCGCCGGTACCGTGGAACTGCACCCGAGTCCAGTCGGCGGCCGGTGTCGCGT
CCGGATGCAGGAGGAAGCCGGCCGGGGTGCCGTCCGCGATGTGCAGGGGTTCGCCACCCGCGAA
GGCCGGCAGTGACGCGCCGGGACCGATGGTGGCGGTGACGTGCTGGAT SEQ ID NO: 35:
CTGGAAACCGTCGTGGCACTGCCCGGCACCGACGGGGTGGCACTGACCGGCCGAATCTCACGTCA GGACGGTCCGGTTCTGTCCGTTGCGGCTTTCGTTGAAATGGCGTTCGCGGCTGCTGGTGGTCGCCC GATCCGTGAACTGTCTGTTGACGCGCTGCTGTACATCCCGGACGACGGCACCGCGGAACTGCAGAC CTGGGTCTCTGAACACCGTCTGACCATCCACGCACGTTACCGTGACACCGAACCGTGGACCCGTCT GGCGACCGCCGCTCTGGACACCACCGCGCCTGCGACGACCCACACCCCGCACCCTGGTCTGATCA CCACGGCGCTGACCCTGACCGGTGACGAAGCACCGGCGATCTGGACTCGGGTGCAGTTCCACGGT ACCGGCGCGGCGACGCTACGGGTCGCGGTGACCCCGGCG
In some embodiments, at least one enoyl reductase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 35-36.
SEQ ID NO: 36:
GAACAGGGCGAGCAACTCGGTCATGATCTCGCGGAGCCGGTCGTGGCCGGCATCCATCAGGGTGA AGGCATGGTAGGTGGCATCCGGGATCTGAGCGGCGTCGCGGATGTCGGTCTGGCCCATCTCGATG AACCGGCCGCCGTCGACCAGCAGGTCGAGGGAGGCGTCGATGAACTC
SEQ ID NO: 37:
GAACAGGGCGAGCAACTCGGTCATGATCTCGCGGAGCCGGTCGTGGCCGGCATCCATCAGGTGGA AGGCATGGTAGGTGGCATCCGGGATCTGAGCGGCGTCGCGGATGTCGGTGCCCATCTCGATGAAC CGGCCGCCGTCGACCAGCAGGTCGAGGGAGGCGTCGATGAACTC
In another aspect, the disclosure provides a nucleic acid encoding any of the foregoing polyketide synthases.
In some embodiments of the invention, the nucleic acid further encodes an LAL, wherein the LAL includes a portion having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the LAL includes a portion having the sequence of SEQ ID NO: 38. In some
embodiments, the LAL has the sequence of SEQ ID NO: 38. In some embodiments, the nucleic acid lacks a TTA regulatory codon in at least one open reading frame.
SEQ ID NO 38: MPAVESYELDARDDELRRLEEAVGQAGNGRGVVVTITGPIACGKTELLDAAAAKSDAITL RAVCSEEERALPYALIGQLIDNPAVASQLPDPVSMALPGEHLSPEAENRLRGDLTRTLLALAAERPVLIGID DMHHADTASLNCLLHLARRVGPARIAMVLTELRRLTPAHSQFHAELLSLGHHREIALRPLGPKHIAELARA GLGPDVDEDVLTGLYRATGGNLNLGHGLIKDVREAWATGGTGINAGRAYRLAYLGSLYRCGPVPLRVAR VAAVLGQSANTTLVRWISGLNADAVGEATEILTEGGLLHDLRFPHPAARSVVLNDLSARERRRLHRSALE VLDDVPVEVVAHHQAGAGFIHGPKAAEIFAKAGQELHVRGELDAASDYLQLAHHASDDAVTRAALRVEAV AIERRRNPLASSRHLDELTVAARAGLLSLEHAALMIRWLALGGRSGEAAEVLAAQRPRAVTDQDRAHLRA AEVSLALVSPGASGVSPGASGPDRRPRPLPPDELANLPKAARLCAIADNAVISALHGRPELASAEAENVL KQADSAADGATALSALTALLYAENTDTAQLWADKLVSETGASNEEEGAGYAGPRAETALRRGDLAAAVE AGSAILDHRRGSLLGITAALPLSSAVAAAIRLGETERAEKWLAEPLPEAIRDSLFGLHLLSARGQYCLATGR HESAYTAFRTCGERMRNWGVDVPGLSLWRVDAAEALLHGRDRDEGRRLIDEQLTHAMGPRSRALTLRV QAAYSPQAQRVDLLEEAADLLLSCNDQYERARVLADLSEAFSALRHHSRARGLLRQARHLAAQCGATPL
LRRLGAKPGGPGWLEESGLPQRIKSLTDAERRVASLAAGGQTNRVIADQLFVTASTVEQHLTNVFRKLGV
KGRQHLPAELANAE. In some embodiments, the nucleic acid further includes an LAL binding site, e.g., an LAL binding site having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%) identity to the sequence of SEQ ID NO: 39 (CTAGGGGGTTGC). In some embodiments, the LAL binding site includes the sequence of SEQ ID NO: 39. In some embodiments, the LAL binding site has the sequence of SEQ ID NO: 39. In some embodiments, the LAL binding site includes the sequence SEQ ID NO: 40
(GGGGGT).
In some embodiments, the nucleic acid further includes an open reading frame positioned such that binding of the LAL to the LAL binding site promotes expression of the open reading frame. In some embodiments, the open reading frame encodes a compound-producing protein (e.g., a polyketide synthase).
In some embodiments, the nucleic acid further encodes a nonribosomal peptide synthase. In some embodiments, the nucleic acid further encodes a first P450 enzyme. In some embodiments, the nucleic acid further encodes a second P450 enzyme.
In some embodiments, the expression vector includes any of the foregoing nucleic acids. In some embodiments, the expression vector is an artificial chromosome (e.g., a bacterial artificial chromosome).
In another aspect, the disclosure provides a host cell including any of the foregoing vectors or polyketide synthases. In some embodiments, the polyketide synthase is heterologous to the host cell.
In some embodiments of the invention, the host cell (e.g., a host cell naturally lacking an LAL and/or an LAL binding site) is engineered to express a recombinant LAL (e.g., a heterologous LAL). In some embodiments, the LAL is constitutively active. In some embodiments, the host cell is engineered by insertion of a LAL binding site in a nucleic acid. In some embodiments, the binding of the recombinant LAL to the LAL binding site promotes transcription of the nucleic acid (e.g., a nucleic acid encoding a compound-producing protein such as a polyketide synthase). In some embodiments, the LAL binding site is heterologous to the LAL. In some embodiments, the LAL binding site is endogenous to the LAL. In some embodiments, the LAL binding site includes the sequence GGGGGT (SEQ ID NO: 40).
In some embodiments, the host cell includes a nucleic acid including a heterologous LAL binding site operably linked to an open reading frame such that binding of an LAL to the heterologous LAL binding site promotes expression of the open reading frame. In some embodiments, the heterologous LAL binding site is a synthetic LAL binding site. In some embodiments, the heterologous LAL binding site promotes greater expression than the endogenous LAL binding site operably linked to the open reading frame. In some embodiments, the heterologous LAL binding site includes at least 8 contiguous nucleotides of C1 -T2-A3-G4-G5-G6-G7-G8-T9-T10-G11 -C12 (SEQ ID NO: 39), wherein none or up to six nucleotides other than any three nucleotides of G4, G5, G6, G7, Gs, T9, and T10 (e.g., G4, G7, and T9; G5, Gs, and T10; or G6, G7, and Gs) are replaced by any other nucleotide.
In some embodiments, the recombinant LAL includes a portion having at least 70% (e.g., at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to the sequence of SEQ ID NO: 38. In some embodiments, the recombinant LAL includes a portion having the sequence of SEQ ID NO: 38. In some embodiments, the recombinant LAL has the amino acid sequence of SEQ ID NO: 38.
In some embodiments, the host cell is a bacterium (e.g., an actinobacterium such as
Streptomyces ambofaciens, Streptomyces hygroscopicus, or Streptomyces malayensis). In some embodiments, the actinobaceterium is S1391 , S1496, or S2441 .
In some embodiments, the host cell has been modified to enhance expression of a compound- producing protein (e.g., a polyketide synthase). For example, in some embodiments, the host cell has been modified to enhance expression of a compound-producing protein (e.g., a polyketide synthase) by (i) deletion of an endogenous gene cluster which expresses a compound-producing protein (e.g., a polyketide synthase); (ii) insertion of a heterologous gene cluster which expresses a compound-producing protein (e.g., a polyketide synthase); (iii) exposure of the host cell to an antibiotic challenge; and/or (iv) introduction of a heterologous promoter that results in at least a two-fold increase in expression of a compound compared to the homologous promoter. An additional method to enhance the expression of polyketides is to optimize media conditions for growth. This includes the specific chemical and nutrient composition of the media, whether the fermentation is conducted in liquid or solid media, the time course of the fermentation, and the volume/scale of the fermentation run.
In another aspect, the disclosure provides a method of producing a polyketide, the method including the step of culturing any of the foregoing host cells under suitable conditions.
In another aspect, the disclosure provides a method of producing a polyketide, the method including the step of culturing a host cell engineered to express any of the foregoing polyketide synthases under conditions suitable for the polyketide synthase to produce a polyketide.
In another aspect, the disclosure provides a method of modulating the activity of a polyketide synthase, the method including the steps of: (a) providing a parent nucleic acid sequence encoding a parent polyketide synthase; and (b) modifying at least one codon of the parent nucleic acid sequence, wherein the codon specifies a residue in a conserved motif of at least one domain of the parent polyketide synthase, wherein the modification results in an alteration of an enzymatic or regulatory activity (e.g., the alteration results in inactivity of the domain) of the at least one domain.
In another aspect, the disclosure provides a method of producing a compound, the method including the steps of: (a) providing a parent nucleic acid encoding a parent polyketide synthase; (b) modifying at least one codon (e.g., a codon in the portion of the nucleic acid which encodes a β-ketone processing domain) of the parent nucleic acid to create a modified nucleic acid encoding a modified polyketide synthase capable of producing a compound, wherein the codon specifies a residue in a conserved domain of at least one domain of the polyketide synthase and wherein the modification results in an alteration of the enzymatic activity of the at least one domain of the polyketide synthase; (c) introducing the modified nucleic acid to a host cell; and (d) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase, thereby producing a compound.
In another aspect, the disclosure provides a method of producing a compound, the method including the steps of: (a) providing a parent polyketide synthase capable of producing a compound; (b) determining the amino acid sequence of the parent polyketide synthase; (c) providing a parent nucleic acid encoding the parent polyketide synthase; (d) modifying at least one codon of the parent nucleic acid to create a modified nucleic acid sequence encoding a modified polyketide synthase capable of producing a compound, wherein the codon specifies a residue in a conserved domain of at least one domain (e.g., a β-ketone processing domain) of the polyketide synthase and wherein the modification results in an alteration of the enzymatic activity (e.g., a decrease in activity) of the at least one domain; (e) introducing the modified nucleic acid to a host cell; (f) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase; and (g) recovering the compound produced by the modified polyketide synthase, thereby producing a compound.
In another aspect, the disclosure provides a method of producing a compound, the method of including the steps of: (a) determining the structure of a parent polyketide synthase; (b) producing a parent nucleic acid encoding the parent polyketide synthase; (c) modifying the nucleic acid to produce a modified nucleic acid encoding a modified polyketide synthase, wherein at least one domain (e.g., a β- ketone processing domain) of the modified polyketide synthase has altered enzymatic activity (e.g., decreased enzymatic activity) compared to the parent polyketide synthase; (d) introducing the modified nucleic acid sequence to a host cell; and (e) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase, thereby producing a compound.
In another aspect, the disclosure provides a method of producing a library of compounds, the method including the steps of: (a) providing a parent nucleic acid sequence encoding a parent polyketide synthase; (b) modifying at least one codon of the parent nucleic acid sequence to create a first modified nucleic acid encoding a first modified polyketide synthase capable of producing a compound; (c) modifying at least one codon of the parent nucleic acid to create a second modified nucleic acid encoding a second modified polyketide synthase capable of producing a compound, wherein the first and second modified nucleic acids are different; (d) introducing the first and the second modified nucleic acid sequences to one or more host cells; and (e) culturing the one or more host cells under conditions suitable to allow expression of a compound by the first and the second modified polyketide synthase, thereby producing a library of compounds.
In another aspect, the disclosure provides a compound produced by any of the foregoing methods.
Definitions
The term "conserved region of a domain," as used herein, refers to the portion of a domain of a polyketide synthase that is substantially the same in all domains of the same type which are active.
As used herein, the term "engineered polyketide synthase" is used to describe a non-natural polyketide synthase whose design and/or production involves action of the hand of man. For example, in some embodiments, an "engineered" polyketide synthase is prepared by production of a non-natural polynucleotide which encodes the polyketide synthase.
A cell that is "engineered to contain" and/or "engineered to express" refers to a cell that has been modified to contain and/or express a protein that does not naturally occur in the cell. A cell may be engineered to contain a protein, e.g., by introducing a nucleic acid encoding the protein by introduction of a vector including the nucleic acid.
The term "functionally inactive," as used herein, refers to a domain of a polyketide synthase that has no activity, or activity below the point of detection. The term "gene cluster that produces a small molecule," as used herein refers to a cluster of genes which encodes one or more compound-producing proteins.
The term "heterologous," as used herein, refers to a relationship between two or more proteins, nucleic acids, compounds, and/or cell that is not present in nature. For example, the LAL having the sequence of SEQ ID NO: 38 is naturally occurring in the S1 8 Streptomyces strain and is thus homologous to that strain and would thus be heterologous to the S12 Streptomyces strain.
The term "homologous," as used herein, refers to a relationship between two or more proteins, nucleic acids, compounds, and/or cells that is present naturally. For example, the LAL having the sequence of SEQ ID NO: 38 is naturally occurring in the S1 8 Streptomyces strain and is thus homologous to that strain.
The term "modified domain," as used herein, refers to a domain of a polyketide synthase in which at least one amino acid residue has been altered from a reference sequence.
A "polyketide synthase" refers to an enzyme belonging to the family of multi-domain enzymes capable of producing a polyketide. A polyketide synthase may be expressed naturally in bacteria, fungi, plants, or animals.
The term "recombinant," as used herein, refers to a protein that is produced using synthetic methods.
As used herein, the term "reference polyketide synthase" refers to a polyketide synthase that has a sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) to the sequence of an engineered polyketide synthase except to the sequence of domains which are modified.
Brief Description of the Drawings
FIG. 1 is an image illustrating the biosynthesis of polyketides by a polyketide synthase.
FIGS. 2A and 2B are images illustrating modification of malonyl β-ketones by domains of polyketide synthases.
FIG. 3 is an image illustrating the biosynthesis of a polyketide.
FIGS. 4A and 4B are sequence alignments illustrating mutations resulting in inactive domains in polyketide synthases.
FIG. 5A is an image illustrating the deactivation of a ketoreductase domain.
FIGS. 5B and 5C are images illustrating the generation of a compound by a modified polyketide synthase.
FIGS. 6A-6C are images illustrating the generation of compounds by modified polyketide synthases.
FIG. 7A is an image of a compound generated by a modified polyketide synthase.
FIG. 7B is a graph illustrating generation of a compound by a modified polyketide synthase.
FIG. 7C is an image illustrating binding of a compound to CEP250.
FIG. 7D is a graph illustrating binding of compounds to CEP250.
FIG. 8A is an image illustrating a target-ID method used for assaying compounds in crude extracts.
FIG. 8B is an image illustrating binding of compounds to CEP250 and CBY1 . FIG. 9A is an image illustrating deactivation of a ketoreductase domain in a polyketide synthase. FIG. 9B is an image illustrating generation of a ring expanded compound by deactivation of a ketoreductase domain in a polyketide synthase.
Detailed Description
The present inventors have discovered that short protein sequences in polyketide synthases that result in deactivated β-ketone processing domains, i.e., ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains, may be grafted onto live domains in another polyketide synthase to deactivate domain activity, and alter the chemical structure of the polyketide produced by the polyketide synthase.
Compounds
Compounds that may be produced with the methods of the invention include, but are not limited to, polyketides and polyketide macrolide antibiotics such as erythromycin ; hybrid polyketides/non- ribosomal peptides such as rapamycin and FK506; carbohydrates including aminoglycoside antibiotics such as gentamicin, kanamycin, neomycin, tobramycin ; benzofuranoids; benzopyranoids; flavonoids; glycopeptides including vancomycin; lipopeptides including daptomycin; tannins; lignans; polycyclic aromatic natural products, terpenoids, steroids, sterols, oxazolidinones including linezolid; amino acids, peptides and peptide antibiotics including polymyxins, non-ribosomal peptides, β-lactams antibiotics including carbapenems, cephalosporins, and penicillin; purines, pteridines, polypyrroles, tetracyclines, quinolones and fluoroquinolones; and sulfonamides.
Proteins
Polyketide Synthases
Polyketide synthases (PKSs) are a family of multi-domain enzymes that produce polyketides. Type I polyketide synthases are large, modular proteins which include several domains organized into modules. The modules generally present in a polyketide synthase include i) a loading module; ii) extending modules; and iii) releasing and/or cyclization modules depending on whether the final polyketide is linear or cyclic. The domains which generally are found in the modules are acyltransferase, acyl carrier protein, keto-synthase, ketoreductase, dehydratase, enoylreductase, methyltransferase, sulfhydrolase, and thioesterase.
A polyketide chain and the starter groups are generally bound to the thiol groups of the active site cysteines in the ketosynthase domain (the polyketide chain) and acyltransferase domain (the loading group and malonyl extender units) through a thioester linkage. Binding to acyl carrier protein (ACP) is mediated by the thiol of the phosphopantetheinyl group, which is bound to a serine hydroxyl of ACP, to form a thioester linkage to the growing polyketide chain. The growing polyketide chain is handed over from one thiol group to another by trans-acylations and is released after synthesis by hydrolysis or cyclization.
The synthesis of a polyketide begins by a starter unit, being loaded onto the acyl carrier protein domain of the PKS catalyzed by the acyltransferase in the loading module. An extender unit, e.g., a malonyl-CoA, is loaded onto the acyl carrier protein domain of the current module catalyzed by another acyltransferase domain. The polyketide chain is then elongated by subsequent extender modules after being passed from the acyl carrier protein domain of module n to the ketosynthase domain of the n+1 module. The acyl carrier protein bound extender unit reacts with the polyketide chain bound to the ketosynthase domain with expulsion of CO2 to produce an extended polyketide chain bound to the acyl carrier protein. Each added extender unit may then be modified by β-ketoprocessing domains, i.e., ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons). Once the synthesis of the polyketide is complete, a thioesterase domain in the releasing modules hydrolyzes the completed polyketide chain from the acyl carrier protein of the last extending module. The compound released from the PKS may then be further modified by other proteins, e.g., nonribosomal peptide synthase. An example of the synthesis of a polyketide by a PKS is illustrated in FIG. 3. In some cases (e.g., rapamycin and X1 , the cluster that encodes Compound 1 ), the biosynthetic cluster harbors polyketide megasynthases and a non-ribosomal peptide synthase (NRPS). This hybrid architecture is referred to as hybrid PKS/NRPS. In the case of rapamycin and Compound 1 , the NRPS module inserts the pipecolate moiety in the FKBP12-binding region of the molecules (FIG. 3). β-ketone processing domains
β-ketone processing domains are the domains in a PKS which result in modification of the elongation groups added during the synthesis of a polyketide. Each β-ketone processing domain is capable of changing the oxidation state of an elongation group. The β-ketone processing domains include ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons).
Non-functional Domains
A comprehensive analysis of β-ketone modifying domains indicated the presence of nonfunctional β-ketone modifying domains which do not affect the final structure of the polyketide. These domains are likely "dead" (FIG. 3). Protein sequence alignments in combination with homology modeling using the crystal structures of functionally related domains as a template revealed that these nonfunctional domains have mutations in key catalytic and substrate binding motifs that render them inactive (FIGS. 4A and 4B). Nevertheless, these "dead" domains are retained in the gene cluster through evolution, suggesting that they instead play a structural role, i.e., maintaining proper spatial organization of the catalytic domains in the module for efficient assembly-line polyketide synthesis. Domain activity may have been selectively turned "off" by evolution, modifying the natural product chemical structure, protein target engagement, and the physiochemical properties of the evolved molecules.
For ketoreductase domain-level engineering, three KR dead domains have been analyzed: KR3 from S9, KR6-S303, and KR3-S399. KR3 from S9 includes single Ala to Glu substitution near the conserved catalytic YAAAN motif. While not being bound by the theory, homology modeling (using PDB 2FRO) suggested that a glutamic acid at this position might form a salt bridge with a nearby arginine, and that the resulting salt bridge would block the mobility of the substrate capping region (aFG) and prevent access of the ketoreductase active site to the polyketide substrate. The S303 and S399 dead KR6 domains include more prominent lesions. In S303, the catalytic Tyr is replaced by Phe and the aFG helix was deleted. In S399, a larger 150 residue deletion encompassing the catalytic and substrate binding residues is present.
In some embodiments, at least one ketoreductase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 12-23.
For dehydratase domain-level engineering, four "dead" DH domains were analyzed: S679-DH7, S12-DH6, S12-DH7, S679-DH4, and S12-DH2. The essential active site residues of the DH domain are distributed across four key conserved motifs: HXXXGXXXXP, GYXYGPXF, DXXX(Q/H) and LPFXW. S679-DH7 has a single Gly to Asp substitution in the HXXXGXXXXP motif, which contains the His residue that deprotonates the polyketide substrate to initiate the dehydration reaction. S12-DH6 and S12- DH7 have substitutions in the LPFXW motif. S679-DH4 contains a significant internal deletion, and S12- DH2 harbors mutations in all four key motifs comprising the DH active site. Hybrid PKS/NRPS clusters such as rapamycin require a hydroxyl for macrocyclization, and thus the S12-DH2 "dead" DH domain must remain inactive for cyclization and biological activity.
In some embodiments, at least one dehydratase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 24-35.
For enoyl-reductase domain level engineering, two ER domains from S12 and S61 were analyzed. Both dead ER domains are located in the loading modules of each cluster and are therefore associated with the chemistry of the starter unit and not the malonyl-derived polyketide chain. In both dead domains, the invariant Lys-Arg dyad is substituted or deleted.
In some embodiments, at least one enoyl reductase domain of a polyketide synthase of the invention is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to SEQ ID No: 36 or 37.
LALs
LALs include three domains, a nucleotide-binding domain, an inducer-binding domain, and a DNA-binding domain. A defining characteristic of the structural class of regulatory proteins that include the LALs is the presence of the AAA+ ATPase domain. Nucleotide hydrolysis is coupled to large conformational changes in the proteins and/or multimerization, and nucleotide binding and hydrolysis represents a "molecular timer" that controls the activity of the LAL (e.g., the duration of the activity of the LAL). The LAL is activated by binding of a small-molecule ligand to the inducer binding site. In most cases the allosteric inducer of the LAL is unknown. In the case of the related protein MalT, the allosteric inducer is maltotriose. Possible inducers for LAL proteins include small molecules found in the environment that trigger compound (e.g., polyketide) biosynthesis. The regulation of the LAL controls production of compound-producing proteins (e.g., polyketide synthases) resulting in activation of compound (e.g., polyketide) production in the presence of external environmental stimuli. Therefore, there are gene clusters that produce small molecules (e.g., PKS gene clusters) which, while present in a strain, do not produce compound either because (i) the LAL has not been activated, (ii) the strain has LAL binding sites that differ from consensus, (iii) the strain lacks an LAL regulator, or (iv) the LAL regulator may be poorly expressed or not expressed under laboratory conditions. Since the DNA binding region of the LALs of the known PKS LALs are highly conserved, the known LALs may be used interchangeably to activate PKS gene clusters other than those which they naturally regulate. In some embodiments, the LAL is a fusion protein.
In some embodiments, an LAL may be modified to include a non-LAL DNA-binding domain, thereby forming a fusion protein including an LAL nucleotide-binding domain and a non-LAL DNA-binding domain. In certain embodiments, the non-LAL DNA-binding domain is capable of binding to a promoter including a protein-binding site positioned such that binding of the DNA-binding domain to the protein- binding site of the promoter promotes expression of a gene of interest (e.g., a gene encoding a compound-producing protein, as described herein). The non-LAL DNA binding domain may include any DNA binding domain known in the art. In some instances, the non-LAL DNA binding domain is a transcription factor DNA binding domain. Examples of non-LAL DNA binding domains include, without limitation, a basic helix-loop-helix (bHLH) domain, leucine zipper domain (e.g., a basic leucine zipper domain), GCC box domain, helix-turn-helix domain, homeodomain, srf-like domain, paired box domain, winged helix domain, zinc finger domain, HMG-box domain, Wor3 domain, OB-fold domain,
immunoglobulin domain, B3 domain, TAL effector domain, Cas9 DNA binding domain, GAL4 DNA binding domain, and any other DNA binding domain known in the art. In some instances, the promoter is positioned upstream to the gene of interest, such that the fusion protein may bind to the promoter and induce or inhibit expression of the gene of interest. In certain instances, the promoter is a heterologous promoter introduced to the nucleic acid (e.g., a chromosome, plasmid, fosmid, or any other nucleic acid construct known in the art) containing the gene of interest. In other instances, the promoter is a preexisting promoter positioned upstream to the gene of interest. The protein-binding site within the promoter may, for example, be a non-LAL protein-binding site. In certain embodiments, the protein- binding site binds to the non-LAL DNA binding domain, thereby forming a cognate DNA binding domain/ protein-binding site pair.
In some embodiments, the LAL is encoded by a nucleic acid having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 41 -62 or has a sequences with at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 63-73.
SEQ ID NO: 41 :
ATGCCTGCCGTGGAGTGCTATGAACTGGACGCCCGCGATGACGAGCTCAGAAAACTGGAGGAGGTT
GTGACCGGGCGGGCCAACGGCCGGGGTGTGGTGGTCACCATCACCGGACCGATCGCCTGCGGCA
AGACCGAACTGCTCGACGCAGCCGCCGCGAAGGCCGACGCCATCACGTTACGAGCGGTCTGCTCC GCGGAGGAACAGGCACTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGGCGCTCGCCTC CCACGCGCTGGAGCCGGCCTGCCCGACCCTCCCGGGCGAGCACCTGTCGCCGGAGGCCGAGAAC CGGCTGCGCAGCGACCTCACCCGTACCCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCGG CATCGACGAGTCACACGCGAACGCTTTGTGTCTGCTCCACCTGGCCCGAAGGGTCGGCTCGGCCC GGATCGCCATGGTCCTCACCGAGTTGCGCCGGCTCACCCCGGCCCACTCACAGTTCCAGGCCGAG CTGCTCAGCCTGGGGCACCACCGCGAGATCGCGCTGCGCCCGCTCAGCCCGAAGCACACCGCCGA GCTGGTCCGCGCCGGTCTCGGTCCCGACGTCGACGAGGACGTGCTCACGGGGTTGTACCGGGCGA CCGGCGGCAACCTGAACCTCACCCGCGGACTGATCAACGATGTGCGGGAGGCCTGGGAGACGGGA GGGACGGGCATCAGCGCGGGCCGCGCGTACCGGCTGGCATACCTCGGTTCCCTCTACCGCTGCGG CCCGGTCCCGTTGCGGGTCGCACGGGTGGCCGCCGTGCTGGGCCAGAGCGCCAACACCACCCTG GTGCGCTGGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCAACCGAGATCCTCACCGAAG GCGGCCTGCTGCACGACCTGCGGTTCCCGCACCCGGCGGCCCGTTCGGTGGTACTCAACGACATG TCCGCCCAGGAACGACGCCGCCTGCACCGGTCCGCTCTGGAAGTGCTGGACGACGTGCCCGTGGA AGTGGTCGCGCACCACCAGGTCGGCGCCGGTCTCCTGCACGGCCCGAAGGCCGCCGAGATATTCG CCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGTTGGACACCGCGTCCGACTATCTGCAACTG GCCCACCAGGCCTCCGACGACGCCGTCACCGGGATGCGGGCCGAGGCCGTGGCGATCGAGCGCC GCCGCAACCCGCTGGCCTCGAGCCGGCACCTCGACGAGCTGACCGTCGTCGCCCGTGCCGGGCT GCTCTTCCCCGAGCACACGGCGCTGATGATCCGCTGGCTGGGCGTCGGCGGGCGGTCCGGCGAG GCAGCCGGGCTGCTGGCCTCGCAGCGCCCCCGTGCGGTCACCGACCAGGACAGGGCCCATATGC GGGCCGCCGAGGTATCGCTCGCGCTGGTCAGCCCCGGCACGTCCGGCCCGGACCGGCGGCCGCG TCCGCTCACGCCGGATGAGCTCGCGAACCTGCCGAAGGCGGCCCGGCTCTGCGCGATCGCCGACA ATGCCGTCATGTCGGCCCTGCGCGGTCGTCCCGAGCTCGCCGCGGCCGAGGCGGAGAACGTCCTG CAGCACGCCGACTCGGCGGCGGCCGGCACCACCGCCCTCGCCGCGCTGACCGCCTTGCTGTACG CGGAGAACACCGACACCGCTCAGCTCTGGGCCGACAAGCTGGTCTCCGAGACCGGGGCGTCGAAC GAGGAGGAGGCGGGCTACGCGGGGCCGCGCGCCGAAGCCGCGTTGCGTCGCGGCGACCTGGCC GCGGCGGTCGAGGCAGGCAGCACCGTTCTGGACCACCGGCGGCTCTCGACGCTCGGCATCACCG CCGCGCTACCGCTGAGCAGCGCGGTGGCCGCCGCCATCCGGCTGGGCGAGACCGAGCGGGCGGA GAAGTGGCTCGCCCAGCCGCTGCCGCAGGCCATCCAGGACGGCCTGTTCGGCCTGCACCTGCTCT CGGCGCGCGGCCAGTACAGCCTCGCCACGGGCCAGCACGAGTCGGCGTACACGGCGTTTCGCAC CTGCGGGGAACGTATGCGGAACTGGGGCGTTGACGTGCCGGGTCTGTCCCTGTGGCGCGTCGACG CCGCCGAGGCGCTGCTGCACGGCCGCGACCGGGACGAGGGCCGACGGCTCGTCGACGAGCAACT CACCCGTGCGATGGGACCCCGTTCCCGCGCCTTGACGCTGCGGGTGCAGGCGGCGTACAGCCCG CCGGCGAAGCGGGTCGACCTGCTCGATGAAGCGGCCGACCTGCTGCTCTCCTGCAACGACCAGTA CGAGCGGGCACGGGTGCTCGCCGACCTGAGCGAGACGTTCAGCGCGCTCCGGCACCACAGCCGG GCGCGGGGACTGCTTCGGCAGGCCCGGCACCTGGCCGCCCAGCGCGGCGCGATACCGCTGCTGC GCCGACTCGGGGCCAAGCCCGGAGGCCCCGGCTGGCTGGAGGAATCCGGCCTGCCGCAGCGGAT CAAGTCGCTGACCGACGCGGAGCGGCGGGTGGCGTCGCTGGCCGCCGGCGGACAGACCAACCGC GTGATCGCCGACCAGCTCTTCGTCACGGCCAGCACGGTGGAGCAGCACCTCACGGACGTCTCCACT GGGTCAAGGCCGCCAGCACCTGCCGCCGAACTCGTCTAG
SEQ ID NO: 42
ATGCCTGCCGTGGAGTGCTATGAACTGGACGCCCGCGATGACGAGCTCAGAAAACTGGAGGAGGTT GTGACCGGGCGGGCCAACGGCCGGGGTGTGGTGGTCACCATCACCGGACCGATCGCCTGCGGCA AGACCGAACTGCTCGACGCAGCCGCCGCGAAGGCCGACGCCATCACGCTGCGAGCGGTCTGCTCC GCGGAGGAACAGGCACTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGGCGCTCGCCTC CCACGCGCTGGAGCCGGCCTGCCCGACCCTCCCGGGCGAGCACCTGTCGCCGGAGGCCGAGAAC CGGCTGCGCAGCGACCTCACCCGTACCCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCGG CATCGACGAGTCACACGCGAACGCTTTGTGTCTGCTCCACCTGGCCCGAAGGGTCGGCTCGGCCC GGATCGCCATGGTCCTCACCGAGTTGCGCCGGCTCACCCCGGCCCACTCACAGTTCCAGGCCGAG CTGCTCAGCCTGGGGCACCACCGCGAGATCGCGCTGCGCCCGCTCAGCCCGAAGCACACCGCCGA GCTGGTCCGCGCCGGTCTCGGTCCCGACGTCGACGAGGACGTGCTCACGGGGTTGTACCGGGCGA CCGGCGGCAACCTGAACCTCACCCGCGGACTGATCAACGATGTGCGGGAGGCCTGGGAGACGGGA GGGACGGGCATCAGCGCGGGCCGCGCGTACCGGCTGGCATACCTCGGTTCCCTCTACCGCTGCGG CCCGGTCCCGTTGCGGGTCGCACGGGTGGCCGCCGTGCTGGGCCAGAGCGCCAACACCACCCTG GTGCGCTGGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCAACCGAGATCCTCACCGAAG GCGGCCTGCTGCACGACCTGCGGTTCCCGCACCCGGCGGCCCGTTCGGTGGTACTCAACGACATG TCCGCCCAGGAACGACGCCGCCTGCACCGGTCCGCTCTGGAAGTGCTGGACGACGTGCCCGTGGA AGTGGTCGCGCACCACCAGGTCGGCGCCGGTCTCCTGCACGGCCCGAAGGCCGCCGAGATATTCG CCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGTTGGACACCGCGTCCGACTATCTGCAACTG GCCCACCAGGCCTCCGACGACGCCGTCACCGGGATGCGGGCCGAGGCCGTGGCGATCGAGCGCC GCCGCAACCCGCTGGCCTCGAGCCGGCACCTCGACGAGCTGACCGTCGTCGCCCGTGCCGGGCT GCTCTTCCCCGAGCACACGGCGCTGATGATCCGCTGGCTGGGCGTCGGCGGGCGGTCCGGCGAG GCAGCCGGGCTGCTGGCCTCGCAGCGCCCCCGTGCGGTCACCGACCAGGACAGGGCCCATATGC GGGCCGCCGAGGTATCGCTCGCGCTGGTCAGCCCCGGCACGTCCGGCCCGGACCGGCGGCCGCG TCCGCTCACGCCGGATGAGCTCGCGAACCTGCCGAAGGCGGCCCGGCTCTGCGCGATCGCCGACA ATGCCGTCATGTCGGCCCTGCGCGGTCGTCCCGAGCTCGCCGCGGCCGAGGCGGAGAACGTCCTG CAGCACGCCGACTCGGCGGCGGCCGGCACCACCGCCCTCGCCGCGCTGACCGCCTTGCTGTACG CGGAGAACACCGACACCGCTCAGCTCTGGGCCGACAAGCTGGTCTCCGAGACCGGGGCGTCGAAC GAGGAGGAGGCGGGCTACGCGGGGCCGCGCGCCGAAGCCGCGTTGCGTCGCGGCGACCTGGCC GCGGCGGTCGAGGCAGGCAGCACCGTTCTGGACCACCGGCGGCTCTCGACGCTCGGCATCACCG CCGCGCTACCGCTGAGCAGCGCGGTGGCCGCCGCCATCCGGCTGGGCGAGACCGAGCGGGCGGA GAAGTGGCTCGCCCAGCCGCTGCCGCAGGCCATCCAGGACGGCCTGTTCGGCCTGCACCTGCTCT CGGCGCGCGGCCAGTACAGCCTCGCCACGGGCCAGCACGAGTCGGCGTACACGGCGTTTCGCAC CTGCGGGGAACGTATGCGGAACTGGGGCGTTGACGTGCCGGGTCTGTCCCTGTGGCGCGTCGACG CCGCCGAGGCGCTGCTGCACGGCCGCGACCGGGACGAGGGCCGACGGCTCGTCGACGAGCAACT CACCCGTGCGATGGGACCCCGTTCCCGCGCCTTGACGCTGCGGGTGCAGGCGGCGTACAGCCCG CCGGCGAAGCGGGTCGACCTGCTCGATGAAGCGGCCGACCTGCTGCTCTCCTGCAACGACCAGTA CGAGCGGGCACGGGTGCTCGCCGACCTGAGCGAGACGTTCAGCGCGCTCCGGCACCACAGCCGG GCGCGGGGACTGCTTCGGCAGGCCCGGCACCTGGCCGCCCAGCGCGGCGCGATACCGCTGCTGC GCCGACTCGGGGCCAAGCCCGGAGGCCCCGGCTGGCTGGAGGAATCCGGCCTGCCGCAGCGGAT CAAGTCGCTGACCGACGCGGAGCGGCGGGTGGCGTCGCTGGCCGCCGGCGGACAGACCAACCGC GTGATCGCCGACCAGCTCTTCGTCACGGCCAGCACGGTGGAGCAGCACCTCACGGACGTCTCCACT GGGTCAAGGCCGCCAGCACCTGCCGCCGAACTCGTCTAG
SEQ ID NO: 43
GTGGTTCCTGAAGTGCGAGCAGCCCCCGACGAACTGATCGCCCGCGATGACGAGCTGAGCCGCCT CCAACGGGCACTCACCAGGGCGGGGAGCGGAAGGGGCGGCGTCGTCGCCATCACCGGGCCCATC GCCAGCGGAAAGACGGCGCTGCTCGACGCCGGAGCGGCCAAGTCCGGCTTCGTCGCACTCCGTGC GGTGTGCTCCTGGGAAGAGCGCACTCTGCCGTACGGGATGCTGGGCCAGCTCTTCGACCATCCCG AACTGGCCGCCCAGGCGCCGGACCTTGCCCACTTCACGGCTTCGTGCGAGAGCCCTCAGGCCGGT ACCGACAACCGCCTGCGGGCCGAGTTCACCCGCACCCTGCTGGCGCTCGCCGCGGACTGGCCCGT CCTGATCGGCATCGACGACGTGCACCACGCCGACGCGGAATCACTGCGCTGTCTGCTCCACCTCGC CCGCCGCATCGGCCCGGCCCGCATCGCGGTCGTACTGACCGAGCTGCGCAGACCGACGCCCGCC GACTCCCGCTTCCAGGCGGAACTGCTGAGCCTGCGCTCCTACCAGGAGATCGCGCTCAGACCGCT CACCGAGGCGCAGACCGGCGAACTCGTACGTCGGCACCTCGGCGCGGAGACCCACGAGGACGTCT CCGCCGATACGTTCCGGGCGACCGGCGGGAACCTGCTCCTCGGGCACGGTTTGATCAATGACATC CGGGAGGCGCGGACAGCGGGACGGCCGGGGGTCGTCGCGGGGCGGGCGTACCGGCTCGCGTAC CTCAGCTCGCTCTACCGCTGCGGCCCGAGCGCGCTGCGTGTCGCCCGGGCGTCCGCCGTGCTCG GCGCGAGCGCCGAAGCCGTGCTCGTCCAGCGGATGACCGGACTGAACAAGGACGCGGTCGAACAG GTCTATGAGCAGCTGAACGAGGGACGGCTGCTGCAGGGCGAGCGGTTTCCGCACCCGGCGGCCC GCTCCATCGTCCTTGACGACCTGTCGGCCCTGGAACGCAGAAACCTGCACGAGTCGGCGCTGGAG CTGCTGCGGGACCACGGCGTGGCCGGCAACGTGCTCGCCCGCCACCAGATCGGCGCCGGCCGGG TGCACGGCGAGGAGGCCGTCGAGCTGTTCACCGGGGCCGCACGGGAGCACCACCTGCGCGGTGA ACTGGACGACGCGGCCGGATACCTGGAACTCGCCCACCGTGCCTCCGACGACCCCGTCACGCGCG CCGCACTACGCGTCGGCGCCGCCGCGATCGAGCGCCTCTGCAATCCGGTACGGGCAGGCCGGCAT CTGCCCGAGCTGCTCACCGCGTCGCGCGCGGGACTGCTCTCCAGCGAGCACGCCGTGTCGCTCGC CGACTGGCTGGCGATGGGCGGGCGCCCGGGCGAGGCGGCCGAGGTCCTCGCGACGCAGCGTCC CGCGGCCGACAGCGAGCAGCACCGCGCACTCCTGCGCAGCGGCGAGTTGTCCCTCGCGCTGGTC CACCCCGGCGCGTGGGATCCGTTGCGCCGGACCGATCGGTTCGCCGCGGGCGGGCTCGGCTCGC TTCCCGGACCCGCCCGGCACCGCGCGGTCGCCGACCAAGCCGTCATCGCGGCGCTGCGTGGACG TCTCGACCGGGCGGACGCCAACGCGGAGAGCGTTCTCCAGCACACCGACGCCACGGCGGACCGG ACCACGGCCATCATGGCGTTGCTGGCCCTGCTCTACGCGGAGAACACCGATGCTGTCCAGTTCTGG GTCGACAAACTGGCCGGTGACGAGGGCACCAGGACACCGGCCGACGAGGCGGTCCACGCGGGGT TCAACGCCGAGATCGCGCTGCGCCGCGGCGACTTGATGAGAGCCGTCGAGTACGGCGAGGCAGCG CTCGGCCACCGGCACCTGCCCACCTGGGGAATGGCCGCCGCTCTGCCGCTGAGCAGCACCGTGGT TGCCGCGATCCGGCTCGGCGACCTCGACAGGGCCGAGCGGTGGCTCGCCGAGCCGCTGCCGCAG CAGACGCCGGAGAGCCTCTTCGGGCTGCACCTGCTCTGGGCCCGCGGGCAGCACCACCTCGCGAC CGGGCGGCACGGGGCGGCGTACACGGCGTTCAGGGAATGCGGCGAGCGGATGCGGCGGTGGGC CGTCGACGTGCCGGGCCTGGCCCTGTGGCGGGTCGACGCCGCCGAATCGCTGCTGCTGCTCGGC CGTGACCGTGCCGAAGGACTGCGGCTCGTCTCCGAGCAGCTGTCCCGGCCGATGCGCCCTCGCGC GCGCGTGCAGACGTTACGGGTACAGGCGGCCTACAGTCCGCCGCCCCAACGGATCGACCTGCTCG AAGAGGCCGCCGACCTGCTGGTCACCTGCAACGACCAGTACGAACTGGCAAACGTACTCAGCGACT TGGCAGAGGCCTCCAGCATGGTCCGGCAGCACAGCAGGGCGCGGGGTCTGCTCCGCCGGGCACG GCACCTCGCCACCCAGTGCGGCGCCGTGCCGCTCCTGCGGCGGCTCGGCGCGGAACCCTCGGAC ATCGGCGGAGCCTGGGACGCGACGCTGGGACAGCGGATCGCGTCACTGACGGAGTCGGAGCGGC GGGTGGCCGCGCTCGCCGCGGTCGGGCGTACGAACAGGGAGATCGCCGAGCAGCTGTTCGTCAC GGCCAGCACGGTGGAACAGCACCTCACGAACGTGTTCCGCAAACTGGCGGTGAAGGGCCGCCAGC AGCTTCCGAAGGAACTGGCCGACGTCGGCGAGCCGGCGGACCGCGACCGCCGGTGCGGGTAG SEQ ID NO: 44
ATGGTTCCTGAAGTGCGAGCAGCCCCCGACGAACTGATCGCCCGCGATGACGAGCTGAGCCGCCT CCAACGGGCACTCACCAGGGCGGGGAGCGGAAGGGGCGGCGTCGTCGCCATCACCGGGCCCATC GCCAGCGGAAAGACGGCGCTGCTCGACGCCGGAGCGGCCAAGTCCGGCTTCGTCGCACTCCGTGC GGTGTGCTCCTGGGAAGAGCGCACTCTGCCGTACGGGATGCTGGGCCAGCTCTTCGACCATCCCG AACTGGCCGCCCAGGCGCCGGACCTTGCCCACTTCACGGCTTCGTGCGAGAGCCCTCAGGCCGGT ACCGACAACCGCCTGCGGGCCGAGTTCACCCGCACCCTGCTGGCGCTCGCCGCGGACTGGCCCGT CCTGATCGGCATCGACGACGTGCACCACGCCGACGCGGAATCACTGCGCTGTCTGCTCCACCTCGC CCGCCGCATCGGCCCGGCCCGCATCGCGGTCGTACTGACCGAGCTGCGCAGACCGACGCCCGCC GACTCCCGCTTCCAGGCGGAACTGCTGAGCCTGCGCTCCTACCAGGAGATCGCGCTCAGACCGCT CACCGAGGCGCAGACCGGCGAACTCGTACGTCGGCACCTCGGCGCGGAGACCCACGAGGACGTCT CCGCCGATACGTTCCGGGCGACCGGCGGGAACCTGCTCCTCGGGCACGGTTTGATCAATGACATC CGGGAGGCGCGGACAGCGGGACGGCCGGGGGTCGTCGCGGGGCGGGCGTACCGGCTCGCGTAC CTCAGCTCGCTCTACCGCTGCGGCCCGAGCGCGCTGCGTGTCGCCCGGGCGTCCGCCGTGCTCG GCGCGAGCGCCGAAGCCGTGCTCGTCCAGCGGATGACCGGACTGAACAAGGACGCGGTCGAACAG GTCTATGAGCAGCTGAACGAGGGACGGCTGCTGCAGGGCGAGCGGTTTCCGCACCCGGCGGCCC GCTCCATCGTCCTTGACGACCTGTCGGCCCTGGAACGCAGAAACCTGCACGAGTCGGCGCTGGAG CTGCTGCGGGACCACGGCGTGGCCGGCAACGTGCTCGCCCGCCACCAGATCGGCGCCGGCCGGG TGCACGGCGAGGAGGCCGTCGAGCTGTTCACCGGGGCCGCACGGGAGCACCACCTGCGCGGTGA ACTGGACGACGCGGCCGGATACCTGGAACTCGCCCACCGTGCCTCCGACGACCCCGTCACGCGCG CCGCACTACGCGTCGGCGCCGCCGCGATCGAGCGCCTCTGCAATCCGGTACGGGCAGGCCGGCAT CTGCCCGAGCTGCTCACCGCGTCGCGCGCGGGACTGCTCTCCAGCGAGCACGCCGTGTCGCTCGC CGACTGGCTGGCGATGGGCGGGCGCCCGGGCGAGGCGGCCGAGGTCCTCGCGACGCAGCGTCC CGCGGCCGACAGCGAGCAGCACCGCGCACTCCTGCGCAGCGGCGAGTTGTCCCTCGCGCTGGTC CACCCCGGCGCGTGGGATCCGTTGCGCCGGACCGATCGGTTCGCCGCGGGCGGGCTCGGCTCGC TTCCCGGACCCGCCCGGCACCGCGCGGTCGCCGACCAAGCCGTCATCGCGGCGCTGCGTGGACG TCTCGACCGGGCGGACGCCAACGCGGAGAGCGTTCTCCAGCACACCGACGCCACGGCGGACCGG ACCACGGCCATCATGGCGTTGCTGGCCCTGCTCTACGCGGAGAACACCGATGCTGTCCAGTTCTGG GTCGACAAACTGGCCGGTGACGAGGGCACCAGGACACCGGCCGACGAGGCGGTCCACGCGGGGT TCAACGCCGAGATCGCGCTGCGCCGCGGCGACTTGATGAGAGCCGTCGAGTACGGCGAGGCAGCG CTCGGCCACCGGCACCTGCCCACCTGGGGAATGGCCGCCGCTCTGCCGCTGAGCAGCACCGTGGT TGCCGCGATCCGGCTCGGCGACCTCGACAGGGCCGAGCGGTGGCTCGCCGAGCCGCTGCCGCAG CAGACGCCGGAGAGCCTCTTCGGGCTGCACCTGCTCTGGGCCCGCGGGCAGCACCACCTCGCGAC CGGGCGGCACGGGGCGGCGTACACGGCGTTCAGGGAATGCGGCGAGCGGATGCGGCGGTGGGC CGTCGACGTGCCGGGCCTGGCCCTGTGGCGGGTCGACGCCGCCGAATCGCTGCTGCTGCTCGGC CGTGACCGTGCCGAAGGACTGCGGCTCGTCTCCGAGCAGCTGTCCCGGCCGATGCGCCCTCGCGC GCGCGTGCAGACGCTGCGGGTACAGGCGGCCTACAGTCCGCCGCCCCAACGGATCGACCTGCTCG AAGAGGCCGCCGACCTGCTGGTCACCTGCAACGACCAGTACGAACTGGCAAACGTACTCAGCGACT TGGCAGAGGCCTCCAGCATGGTCCGGCAGCACAGCAGGGCGCGGGGTCTGCTCCGCCGGGCACG GCACCTCGCCACCCAGTGCGGCGCCGTGCCGCTCCTGCGGCGGCTCGGCGCGGAACCCTCGGAC ATCGGCGGAGCCTGGGACGCGACGCTGGGACAGCGGATCGCGTCACTGACGGAGTCGGAGCGGC GGGTGGCCGCGCTCGCCGCGGTCGGGCGTACGAACAGGGAGATCGCCGAGCAGCTGTTCGTCAC
GGCCAGCACGGTGGAACAGCACCTCACGAACGTGTTCCGCAAACTGGCGGTGAAGGGCCGCCAGC
AGCTTCCGAAGGAACTGGCCGACGTCGGCGAGCCGGCGGACCGCGACCGCCGGTGCGGGTAG SEQ ID NO: 45
GTGATAGCGCGCTTATCTCCCCCAGACCTGATCGCCCGCGATGACGAGTTCGGTTCCCTCCACCGG GCGCTCACCCGAGCGGGGGGCGGGCGGGGCGTCGTCGCCGCCGTCACCGGGCCGATCGCCTGC GGCAAGACCGAACTCCTCGACGCCGCCGCGGCCAAGGCCGGCTTCGTCACCCTTCGCGCGGTGTG CTCCATGGAGGAGCGGGCCCTGCCGTACGGCATGCTCGGCCAGCTCCTCGACCAGCCCGAGCTGG CCGCCCGGACACCGGAGCTGGTCCGGCTGACGGCATCGTGCGAAAACCTGCCGGCCGACGTCGAC AACCGCCTGGGGACCGAACTCACCCGCACGGTGCTGACGCTCGCCGCGGAGCGGCCCGTACTGAT CGGCATCGACGACGTGCACCACGCCGACGCGCCGTCGCTGCGCTGCCTGCTCCACCTCGCGCGCC GCATCAGCCGGGCCCGTGTCGCCATCGTGCTGACCGAGCTGCTCCGGCCGACGCCCGCCCACTCC CAATTCCGGGCGGCACTGCTGAGTCTGCGCCACTACCAGGAGATCGCGCTGCGCCCGCTCACCGA GGCGCAGACCACCGAACTCGTGCGCCGGCACCTCGGCCAGGACGCGCACGACGACGTGGTGGCC CAGGCGTTCCGGGCGACCGGCGGCAACCTGCTCCTCGGCCACGGCCTGATCGACGACATCCGGGA GGCACGGACACGGACCTCAGGGTGCCTGGAAGTGGTCGCGGGGCGGGCGTACCGGCTCGCCTAC CTCGGGTCGCTCTATCGTTGCGGCCCGGCCGCGCTGAGCGTCGCCCGAGCTTCCGCCGTGCTCGG CGAGAGTGTCGAACTCACCCTCGTCCAGCGGATGACCGGCCTCGACACCGAGGCGGTCGAGCAGG CCCACGAACAGCTGGTCGAGGGGCGGCTGCTGCGGGAAGGGCGGTTCCCGCACCCCGCGGCCCG CTCCGTCGTACTCGACGACCTCTCCGCCGCCGAGCGGCGTGGCCTGCACGAGCTGGCGCTGGAAC TGCTGCGGGACCGCGGCGTGGCCAGCAAGGTGCTCGCCCGCCACCAGATGGGTACCGGCCGGGT GCACGGCGCCGAGGTCGCCGGGCTGTTCACCGACGCCGCGCGCGAGCACCACCTGCGCGGCGAG CTCGACGAGGCCGTCACCTACCTGGAGTTCGCCTACCGGGCCTCCGACGACCCCGCCGTCCACGC CGCACTGCGCGTCGACACCGCCGCCATCGAGCGGCTCTGCGATCCCGCCAGATCCGGCCGGCATG TGCCCGAGCTGCTCACCGCGTCGCGGGAACGGCTCCTCTCCAGCGAGCACGCCGTGTCGCTCGCC TGCTGGCTGGCGATGGACGGGCGGCCGGGCGAGGCCGCCGAGGTCCTGGCGGCCCAGCGCTCC GCCGCCCCGAGCGAGCAGGGCCGGGCGCACCTGCGCGTCGCGGACCTGTCCCTCGCGCTGATCT ATCCCGGCGCGGCCGATCCGCCGCGTCCGGCCGATCCGCCGGCCGAGGACGAGGTCGCCTCGTT TTCCGGAGCCGTCCGGCACCGCGCCGTCGCCGACAAGGCCCTGAGCAACGCGCTGCGCGGCTGG TCCGAACAGGCCGAGGCCAAAGCCGAGTACGTGCTCCAGCACTCCCGGGTCACGACGGACCGGAC CACGACCATGATGGCGTTGCTGGCCCTGCTCTACGCCGAGGACACCGATGCCGTCCAGTCCTGGGT CGACAAGCTGGCCGGTGACGACAACATGCGGACCCCGGCCGACGAGGCGGTCCACGCGGGGTTC CGCGCCGAGGCCGCGCTGCGCCGCGGCGACCTGACCGCCGCCGTCGAATGCGGCGAGGCCGCG CTCGCCCCCCGGGTCGTGCCCTCCTGGGGGATGGCCGCCGCATTGCCGCTGAGCAGCACCGTGG CCGCCGCGATCCGACTGGGCGACCTGGACCGGGCGGAGCGGTGGCTCGCCGAGCCGTTGCCGGA GGAGACCTCCGACAGCCTCTTCGGACTGCACATGGTCTGGGCCCGTGGGCAACACCATCTCGCGG CCGGGCGGTACCGGGCGGCGTACAACGCGTTCCGGGACTGCGGGGAGCGGATGCGACGCTGGTC CGTCGACGTGCCGGGCCTGGCCCTGTGGCGGGTCGACGCCGCCGAAGCGCTTCTGCTGCTCGGC CGCGGCCGTGACGAGGGGCTGAGGCTCATCTCCGAGCAGCTGTCCCGGCCGATGGGGTCCCGGG CGCGGGTGATGACGCTGCGGGTGCAGGCGGCCTACAGTCCGCCGGCCAAGCGGATCGAACTGCTC GACGAGGCCGCCGATCTGCTCATCATGTGCCGCGACCAGTACGAGCTGGCCCGCGTCCTCGCCGA CATGGGCGAAGCGTGCGGCATGCTCCGGCGGCACAGCCGTGCGCGGGGACTGTTCCGCCGCGCA CGGCACCTCGCGACCCAGTGCGGAGCCGTGCCGCTCCTCCGGCGGCTCGGTGGGGAGTCCTCGG ACGCGGACGGCACCCAGGACGTGACGCCGGCGCAGCGGATCACATCGCTGACCGAGGCGGAGCG GCGGGTGGCGTCGCACGCCGCGGTCGGGCGCACCAACAAGGAGATCGCCAGCCAGCTGTTCGTCA CCTCCAGCACGGTGGAACAGCACCTCACCAACGTGTTCCGCAAGCTGGGGGTGAAGGGCCGTCAG CAACTGCCCAAGGAACTGTCCGACGCCGGCTGA
SEQ ID NO: 46
ATGATAGCGCGCCTGTCTCCCCCAGACCTGATCGCCCGCGATGACGAGTTCGGTTCCCTCCACCGG GCGCTCACCCGAGCGGGGGGCGGGCGGGGCGTCGTCGCCGCCGTCACCGGGCCGATCGCCTGC GGCAAGACCGAACTCCTCGACGCCGCCGCGGCCAAGGCCGGCTTCGTCACCCTTCGCGCGGTGTG CTCCATGGAGGAGCGGGCCCTGCCGTACGGCATGCTCGGCCAGCTCCTCGACCAGCCCGAGCTGG CCGCCCGGACACCGGAGCTGGTCCGGCTGACGGCATCGTGCGAAAACCTGCCGGCCGACGTCGAC AACCGCCTGGGGACCGAACTCACCCGCACGGTGCTGACGCTCGCCGCGGAGCGGCCCGTACTGAT CGGCATCGACGACGTGCACCACGCCGACGCGCCGTCGCTGCGCTGCCTGCTCCACCTCGCGCGCC GCATCAGCCGGGCCCGTGTCGCCATCGTGCTGACCGAGCTGCTCCGGCCGACGCCCGCCCACTCC CAATTCCGGGCGGCACTGCTGAGTCTGCGCCACTACCAGGAGATCGCGCTGCGCCCGCTCACCGA GGCGCAGACCACCGAACTCGTGCGCCGGCACCTCGGCCAGGACGCGCACGACGACGTGGTGGCC CAGGCGTTCCGGGCGACCGGCGGCAACCTGCTCCTCGGCCACGGCCTGATCGACGACATCCGGGA GGCACGGACACGGACCTCAGGGTGCCTGGAAGTGGTCGCGGGGCGGGCGTACCGGCTCGCCTAC CTCGGGTCGCTCTATCGTTGCGGCCCGGCCGCGCTGAGCGTCGCCCGAGCTTCCGCCGTGCTCGG CGAGAGTGTCGAACTCACCCTCGTCCAGCGGATGACCGGCCTCGACACCGAGGCGGTCGAGCAGG CCCACGAACAGCTGGTCGAGGGGCGGCTGCTGCGGGAAGGGCGGTTCCCGCACCCCGCGGCCCG CTCCGTCGTACTCGACGACCTCTCCGCCGCCGAGCGGCGTGGCCTGCACGAGCTGGCGCTGGAAC TGCTGCGGGACCGCGGCGTGGCCAGCAAGGTGCTCGCCCGCCACCAGATGGGTACCGGCCGGGT GCACGGCGCCGAGGTCGCCGGGCTGTTCACCGACGCCGCGCGCGAGCACCACCTGCGCGGCGAG CTCGACGAGGCCGTCACCTACCTGGAGTTCGCCTACCGGGCCTCCGACGACCCCGCCGTCCACGC CGCACTGCGCGTCGACACCGCCGCCATCGAGCGGCTCTGCGATCCCGCCAGATCCGGCCGGCATG TGCCCGAGCTGCTCACCGCGTCGCGGGAACGGCTCCTCTCCAGCGAGCACGCCGTGTCGCTCGCC TGCTGGCTGGCGATGGACGGGCGGCCGGGCGAGGCCGCCGAGGTCCTGGCGGCCCAGCGCTCC GCCGCCCCGAGCGAGCAGGGCCGGGCGCACCTGCGCGTCGCGGACCTGTCCCTCGCGCTGATCT ATCCCGGCGCGGCCGATCCGCCGCGTCCGGCCGATCCGCCGGCCGAGGACGAGGTCGCCTCGTT TTCCGGAGCCGTCCGGCACCGCGCCGTCGCCGACAAGGCCCTGAGCAACGCGCTGCGCGGCTGG TCCGAACAGGCCGAGGCCAAAGCCGAGTACGTGCTCCAGCACTCCCGGGTCACGACGGACCGGAC CACGACCATGATGGCGTTGCTGGCCCTGCTCTACGCCGAGGACACCGATGCCGTCCAGTCCTGGGT CGACAAGCTGGCCGGTGACGACAACATGCGGACCCCGGCCGACGAGGCGGTCCACGCGGGGTTC CGCGCCGAGGCCGCGCTGCGCCGCGGCGACCTGACCGCCGCCGTCGAATGCGGCGAGGCCGCG CTCGCCCCCCGGGTCGTGCCCTCCTGGGGGATGGCCGCCGCATTGCCGCTGAGCAGCACCGTGG CCGCCGCGATCCGACTGGGCGACCTGGACCGGGCGGAGCGGTGGCTCGCCGAGCCGTTGCCGGA GGAGACCTCCGACAGCCTCTTCGGACTGCACATGGTCTGGGCCCGTGGGCAACACCATCTCGCGG CCGGGCGGTACCGGGCGGCGTACAACGCGTTCCGGGACTGCGGGGAGCGGATGCGACGCTGGTC CGTCGACGTGCCGGGCCTGGCCCTGTGGCGGGTCGACGCCGCCGAAGCGCTTCTGCTGCTCGGC CGCGGCCGTGACGAGGGGCTGAGGCTCATCTCCGAGCAGCTGTCCCGGCCGATGGGGTCCCGGG CGCGGGTGATGACGCTGCGGGTGCAGGCGGCCTACAGTCCGCCGGCCAAGCGGATCGAACTGCTC GACGAGGCCGCCGATCTGCTCATCATGTGCCGCGACCAGTACGAGCTGGCCCGCGTCCTCGCCGA CATGGGCGAAGCGTGCGGCATGCTCCGGCGGCACAGCCGTGCGCGGGGACTGTTCCGCCGCGCA CGGCACCTCGCGACCCAGTGCGGAGCCGTGCCGCTCCTCCGGCGGCTCGGTGGGGAGTCCTCGG ACGCGGACGGCACCCAGGACGTGACGCCGGCGCAGCGGATCACATCGCTGACCGAGGCGGAGCG GCGGGTGGCGTCGCACGCCGCGGTCGGGCGCACCAACAAGGAGATCGCCAGCCAGCTGTTCGTCA CCTCCAGCACGGTGGAACAGCACCTCACCAACGTGTTCCGCAAGCTGGGGGTGAAGGGCCGTCAG CAACTGCCCAAGGAACTGTCCGACGCCGGCTGA
SEQ ID NO: 47
GTGGAGTTTTACGACCTGGTCGCCCGCGATGACGAGCTCAGAAGGTTGGACCAGGCCCTCGGCCG CGCCGCCGGCGGACGGGGTGTCGTGGTCACCGTCACCGGACCGGTCGGCTGCGGCAAGACCGAA CTGCTGGACGCGGCCGCGGCCGAGGAGGAATTCATCACGTTGCGTGCGGTCTGCTCGGCCGAGGA GCGGGCCCTGCCGTACGCCGTGATCGGCCAACTCCTCGACCATCCCGTACTCTCCGCACGCGCGC CCGACCTGGCCTGCGTGACGGCTCCGGGCCGGACGCTGCCGGCCGACACCGAGAACCGCCTGCG CCGCGACCTCACCCGGGCCCTGCTGGCCCTGGCCTCCGAACGACCGGTTCTGATCTGCATCGACG ACGTGCACCAGGCCGACACCGCCTCGCTGAACTGCCTGCTGCACCTGGCCCGGCGGGTCGCCTCG GCCCGGATCGCCATGATCCTCACCGAGTTGCGCCGGCTCACCCCGGCTCACTCCCGGTTCGAGGC GGAACTGCTCAGCCTGCGGCACCGCCACGAGATCGCGCTGCGTCCCCTCGGCCCGGCCGACACCG CCGAACTGGCCCGCGCCCGGCTCGGCGCCGGCGTCACCGCCGACGAGCTGGCCCAGGTCCACGA GGCCACCAGCGGGAACCCCAACCTGGTCGGAGGCCTGGTCAACGACGTGCGAGAGGCCTGGGCG GCCGGTGGCACGGGCATTGCGGCGGGGCGGGCGTACCGGCTGGCGTACCTCAGCTCCGTGTACC GCTGTGGTCCGGTCCCGTTGCGGATCGCCCAGGCGGCGGCGGTGCTGGGTCCCAGCGCCACCGT CACGCTGGTGCGCCGGATCAGCGGGCTCGACGCCGAGACGGTGGACGAGGCGACCGCGATCCTC ACCGAGGGCGGCCTGCTCCGGGACCACCGGTTCCCGCATCCGGCGGCCCGCTCGGTCGTACTCGA CGACATGTCCGCGCAGGAACGCCGCCGCCTGCACCGGTCCACGCTGGACGTGCTGGACGGCGTAC CCGTCGACGTGCTCGCGCACCACCAGGCCGGCGCCGGTCTGCTGCACGGCCCGCAGGCGGCCGA GATGTTCGCCCGGGCCAGCCAGGAGCTGCGGGTACGCGGCGAGCTGGACGCCGCGACCGAGTAC CTGCAACTGGCCTACCGGGCCTCCGACGACGCCGGCGCCCGGGCCGCCCTGCAGGTGGAGACCG TGGCCGGCGAGCGCCGCCGCAACCCGCTGGCCGCCAGCCGGCACCTGGACGAGCTGGCCGCCGC CGCCCGGGCCGGCCTGCTGTCGGCCGAGCACGCCGCCCTGGTCGTGCACTGGCTGGCCGACGCC GGACGACCCGGCGAGGCCGCCGAGGTGCTGGCGCTGCAGCGGGCGCTGGCCGTCACCGACCACG ACCGGGCCCGCCTGCGGGCGGCCGAGGTGTCGCTCGCGCTGTTCCACCCCGGCGTCCCCGGTTC GGACCCGCGGCCCCTCGCGCCGGAGGAGCTCGCGAGCCTGTCCCTGTCGGCCCGGCACGGTGTG ACCGCCGACAACGCGGTGCTGGCGGCGCTGCGCGGCCGTCCCGAGTCGGCCGCCGCCGAGGCG GAGAACGTGCTGCGCAACGCCGACGCCGCCGCGTCCGGCCCGACCGCCCTGGCCGCGCTGACGG CCCTGCTCTACGCCGAGAACACCGACGCCGCCCAGCTCTGGGCGGACAAGCTGGCCGCGGGCATC GGGGCGGGGGAGGGGGAGGCCGGCTACGCGGGGCCGCGGACCGTGGCCGCCCTGCGTCGCGGC GACCTGACCACCGCGGTCCAGGCGGCCGGCGCGGTCCTGGACCGCGGCCGGCCGTCGTCGCTCG GCATCACCGCCGTGTTGCCGTTGAGCGGCGCGGTCGCCGCCGCGATCCGGCTGGGCGAGCTCGA GCGGGCCGAGAAGTGGCTGGCCGAGCCGCTGCCCGAAGCCGTCCACGACAGCCTGTTCGGCCTG CACCTGCTGATGGCGCGGGGCCGCTACAGCCTCGCGGTGGGCCGGCACGAGGCGGCGTACGCCG CGTTCCGGGACTGCGGTGAACGGATGCGCCGGTGGGACGTCGACGTGCCCGGGCTGGCCCTGTG GCGGGTGGACGCGGCCGAGGCGCTGCTGCCCGGCGATGACCGGGCGGAGGGCCGGCGGCTGAT CGACGAGCAGCTCACCCGGCCGATGGGGCCCCGGTCACGAGCCCTGACCCTGCGGGTACGAGCG GCCTACGCCCCGCCGGCGAAACGGATCGACCTGCTCGACGAAGCGGCCGACCTGCTGCTCTCCAG CAACGACCAGTACGAGCGGGCACGGGTGCTGGCCGACCTGAGCGAGGCGTTCAGCGCGCTCCGG CAGAACGGCCGGGCGCGCGGCATCCTGCGGCAGGCCCGGCACCTGGCCGCCCAGTGCGGGGCG GTCCCCCTGCTGCGCCGGCTGGGCGTCAAGGCCGGCCGGTCCGGTCGGCTCGGCCGGCCGCCGC AGGGAATCCGCTCCCTGACCGAGGCCGAGCGCCGGGTGGCCACGCTGGCCGCCGCCGGGCAGAC CAACCGGGAGATCGCCGACCAGCTCTTCGTCACCGCCAGCACGGTCGAGCAGCACCTCACCAACG TGTTCCGCAAGCTCGGCGTGAAGGGCCGCCAGCAATTGCCGGCCGAGCTGGCCGACCTGCGGCCG CCGGGCTGA
SEQ ID NO: 48
ATGGAGTTTTACGACCTGGTCGCCCGCGATGACGAGCTCAGAAGGTTGGACCAGGCCCTCGGCCG CGCCGCCGGCGGACGGGGTGTCGTGGTCACCGTCACCGGACCGGTCGGCTGCGGCAAGACCGAA CTGCTGGACGCGGCCGCGGCCGAGGAGGAATTCATCACGTTGCGTGCGGTCTGCTCGGCCGAGGA GCGGGCCCTGCCGTACGCCGTGATCGGCCAACTCCTCGACCATCCCGTACTCTCCGCACGCGCGC CCGACCTGGCCTGCGTGACGGCTCCGGGCCGGACGCTGCCGGCCGACACCGAGAACCGCCTGCG CCGCGACCTCACCCGGGCCCTGCTGGCCCTGGCCTCCGAACGACCGGTTCTGATCTGCATCGACG ACGTGCACCAGGCCGACACCGCCTCGCTGAACTGCCTGCTGCACCTGGCCCGGCGGGTCGCCTCG GCCCGGATCGCCATGATCCTCACCGAGTTGCGCCGGCTCACCCCGGCTCACTCCCGGTTCGAGGC GGAACTGCTCAGCCTGCGGCACCGCCACGAGATCGCGCTGCGTCCCCTCGGCCCGGCCGACACCG CCGAACTGGCCCGCGCCCGGCTCGGCGCCGGCGTCACCGCCGACGAGCTGGCCCAGGTCCACGA GGCCACCAGCGGGAACCCCAACCTGGTCGGAGGCCTGGTCAACGACGTGCGAGAGGCCTGGGCG GCCGGTGGCACGGGCATTGCGGCGGGGCGGGCGTACCGGCTGGCGTACCTCAGCTCCGTGTACC GCTGTGGTCCGGTCCCGTTGCGGATCGCCCAGGCGGCGGCGGTGCTGGGTCCCAGCGCCACCGT CACGCTGGTGCGCCGGATCAGCGGGCTCGACGCCGAGACGGTGGACGAGGCGACCGCGATCCTC ACCGAGGGCGGCCTGCTCCGGGACCACCGGTTCCCGCATCCGGCGGCCCGCTCGGTCGTACTCGA CGACATGTCCGCGCAGGAACGCCGCCGCCTGCACCGGTCCACGCTGGACGTGCTGGACGGCGTAC CCGTCGACGTGCTCGCGCACCACCAGGCCGGCGCCGGTCTGCTGCACGGCCCGCAGGCGGCCGA GATGTTCGCCCGGGCCAGCCAGGAGCTGCGGGTACGCGGCGAGCTGGACGCCGCGACCGAGTAC CTGCAACTGGCCTACCGGGCCTCCGACGACGCCGGCGCCCGGGCCGCCCTGCAGGTGGAGACCG TGGCCGGCGAGCGCCGCCGCAACCCGCTGGCCGCCAGCCGGCACCTGGACGAGCTGGCCGCCGC CGCCCGGGCCGGCCTGCTGTCGGCCGAGCACGCCGCCCTGGTCGTGCACTGGCTGGCCGACGCC GGACGACCCGGCGAGGCCGCCGAGGTGCTGGCGCTGCAGCGGGCGCTGGCCGTCACCGACCACG ACCGGGCCCGCCTGCGGGCGGCCGAGGTGTCGCTCGCGCTGTTCCACCCCGGCGTCCCCGGTTC GGACCCGCGGCCCCTCGCGCCGGAGGAGCTCGCGAGCCTGTCCCTGTCGGCCCGGCACGGTGTG ACCGCCGACAACGCGGTGCTGGCGGCGCTGCGCGGCCGTCCCGAGTCGGCCGCCGCCGAGGCG GAGAACGTGCTGCGCAACGCCGACGCCGCCGCGTCCGGCCCGACCGCCCTGGCCGCGCTGACGG CCCTGCTCTACGCCGAGAACACCGACGCCGCCCAGCTCTGGGCGGACAAGCTGGCCGCGGGCATC GGGGCGGGGGAGGGGGAGGCCGGCTACGCGGGGCCGCGGACCGTGGCCGCCCTGCGTCGCGGC GACCTGACCACCGCGGTCCAGGCGGCCGGCGCGGTCCTGGACCGCGGCCGGCCGTCGTCGCTCG GCATCACCGCCGTGTTGCCGTTGAGCGGCGCGGTCGCCGCCGCGATCCGGCTGGGCGAGCTCGA GCGGGCCGAGAAGTGGCTGGCCGAGCCGCTGCCCGAAGCCGTCCACGACAGCCTGTTCGGCCTG CACCTGCTGATGGCGCGGGGCCGCTACAGCCTCGCGGTGGGCCGGCACGAGGCGGCGTACGCCG CGTTCCGGGACTGCGGTGAACGGATGCGCCGGTGGGACGTCGACGTGCCCGGGCTGGCCCTGTG GCGGGTGGACGCGGCCGAGGCGCTGCTGCCCGGCGATGACCGGGCGGAGGGCCGGCGGCTGAT CGACGAGCAGCTCACCCGGCCGATGGGGCCCCGGTCACGAGCCCTGACCCTGCGGGTACGAGCG GCCTACGCCCCGCCGGCGAAACGGATCGACCTGCTCGACGAAGCGGCCGACCTGCTGCTCTCCAG CAACGACCAGTACGAGCGGGCACGGGTGCTGGCCGACCTGAGCGAGGCGTTCAGCGCGCTCCGG CAGAACGGCCGGGCGCGCGGCATCCTGCGGCAGGCCCGGCACCTGGCCGCCCAGTGCGGGGCG GTCCCCCTGCTGCGCCGGCTGGGCGTCAAGGCCGGCCGGTCCGGTCGGCTCGGCCGGCCGCCGC AGGGAATCCGCTCCCTGACCGAGGCCGAGCGCCGGGTGGCCACGCTGGCCGCCGCCGGGCAGAC CAACCGGGAGATCGCCGACCAGCTCTTCGTCACCGCCAGCACGGTCGAGCAGCACCTCACCAACG TGTTCCGCAAGCTCGGCGTGAAGGGCCGCCAGCAATTGCCGGCCGAGCTGGCCGACCTGCGGCCG CCGGGCTGA
SEQ ID NO: 49
GTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAA GGCTGAGGCCATCATTCTGCGCGCGGTCTGCGCGCCAGAAGAGCGGGCTATGCCGTACGCCATGA TCGGGCAGCTCATCGACGACCCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGC CCAGGGCGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTG CTGGCGCTTGCCGTCGACCGGCCTGTGCTGATCGGCGTCGACGATGTGCATCACGCCGACACCGC CTCTTTGAACTGTCTGCTGCATTTGGCGCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACC GAGTTGCGCAGCCTCACCCCTACTCAGTCACGGTTCAAGGCGGAGCTGCTCAGCCTGCCGTACCAC CACGAGATCGCGCTGCGTCCGTTCGGACCGGAGCAATCGGCGGAGCTGGCCCGCGCCGCCTTCG GCCCGGGCCTCGCCGAGGATGTGCTCGTGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCA GCCGTGGACTGATCAGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCCTTCGAGGCGGG CCGCGCGTTCCGGCTGGCGTACCTCGGCTCGCTCTACCGCTGTGGCCCGGTCGCGCTGCGGGTCG CCCGAGTGGCTGCCGTGCTGGGCCCGAGCGCCACCACCACGCTGGTGCGCCGTCTAAGCGGGCT CAGCGCGGAGACGATAGACCGGGCAACCAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGC AGTTCCCGCACCCGGCCGCCCGCTCGGTGGTGCTTGATGACATGTCCGCCCAGGAACGACGCGGC CTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGT CGGCGCCGGTCTCATACACGGGCCCAAGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGG TCGTACGCAACGAGTTGGGCGACGCGGCAGAATACCTGCAACTGGCTCACCGGGCCTCCGACGAT GTCTCCACCCGGGCCGCCTTACGGGTCGAGGCCGTGGCGATCGAGCGCCGCCGCAATCCGCTGG CCTCCAGTCGGCACATGGACGAGCTGAGCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCAT GCGGCGCTGGCCGTCTTCTGGCTGGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCTGG CGTCGGAACGCCCGCTAGCGACCACCGATCAGAACCGGGCCCACTTGCGATTTGTCGAGGTGACTC TCGCGCTGTTCTCTCCCGGCGCCTTCGGATCGGACCGGCGCCCACCTCCGCTGACGCCGGACGAA CTCGCCAGCCTGCCGAAGGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTT GCACGGTCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAGC CGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTACGCGGAGAACACCGAGTCCGCTCATATCTG GGCCGACAAGCTGGGCAGCACGAATGGCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGC CCGTGCGCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCACCG TCCTGGACGACCGGTCGCTGCCGTCGCTCGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGACG GCCGCCGCTGTCCGGCTGGGCGAACTCGAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAA CGGCGTCCAGGACAGCCTTTTCGGTCTGCACCTGCTCTCGGCATACGGCCAGTACAGCCTCGCGAT GGGCCGATATGAATCGGCTCTCCGGGCGTTTCACACCTGCGGAGAACGTATGCGCAGCTGGGATGT TGACGTGCCTGGTCTGGCCCTGTGGCGTGTCGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGA ACGAGGGCCAGCGGCTCATCGACGAACAACTCACCCGTCCGATGGGGCCTCGTTCCCGCGCGTTA ACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCGGC CGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGACA CGCTCAGCGCGCTCAGACGCTATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCC GCCCAGTGCGGTGCTGTCCCGCTGCTGCGCAGGCTCGGGGGCGAGCCCGGCCGGATCGACGACG CCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAGCGGCGGGTGGCGGCGCTGGCCGC GGCCGGACAGACCAACCGGGAGATCGCCAAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGC ACCTCACAAGCGTCTTCCGCAAACTGGGGGTCAAGGGTCGCAAGCAGCTGCCGACCGCGCTGGCC GACGTGGAACAGACCTGA
SEQ ID NO: 50
ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGACGAACTCGGCATTCTA CAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGGGCGTCGTGGTCACCGTCACCGGCCCAATCGC CTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGG TCTGCGCGCCAGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGACCCGGCG CTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCCCAGGGCGGGCAGCTGTCGCTGAGGG CCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTGCTGGCGCTTGCCGTCGACCGGCCTGTG CTGATCGGCGTCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATTTGGCG CGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCAGCCTCACCCCTACTCAG TCACGGTTCAAGGCGGAGCTGCTCAGCCTGCCGTACCACCACGAGATCGCGCTGCGTCCGTTCGG ACCGGAGCAATCGGCGGAGCTGGCCCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCTC GTGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGACTGATCAGCGATGTGCGG GAGGCCCTGGCCAACGGAGAGAGCGCCTTCGAGGCGGGCCGCGCGTTCCGGCTGGCGTACCTCG GCTCGCTCTACCGCTGTGGCCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCC GAGCGCCACCACCACGCTGGTGCGCCGTCTAAGCGGGCTCAGCGCGGAGACGATAGACCGGGCAA CCAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGCAGTTCCCGCACCCGGCCGCCCGCTCG GTGGTGCTTGATGACATGTCCGCCCAGGAACGACGCGGCCTGCACACTCTCGCCCTGGAACTGCTG GACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCAA GGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTACGCAACGAGTTGGGCGACGCGG CAGAATACCTGCAACTGGCTCACCGGGCCTCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGTC GAGGCCGTGGCGATCGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAGCTGAG CGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCCGTCTTCTGGCTGGCCG ACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCTGGCGTCGGAACGCCCGCTAGCGACCACCGA TCAGAACCGGGCCCACTTGCGATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGG ATCGGACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAAGGCGGCCTGG CAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGCACGGTCATCCAGAACTTGCCACCGCT CAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGC CCTGTTGTACGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCACGAATGG CGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGAGATCGCCCTGCGGCGC GGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCACCGTCCTGGACGACCGGTCGCTGCCGTCGCT CGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTCG AGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGACAGCCTTTTCGGTCTG CACCTGCTCTCGGCATACGGCCAGTACAGCCTCGCGATGGGCCGATATGAATCGGCTCTCCGGGC GTTTCACACCTGCGGAGAACGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGCG TGTCGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATCGACGAAC AACTCACCCGTCCGATGGGGCCTCGTTCCCGCGCGCTGACGCTGCGGATCAAGGCGGCATACCTC CCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCC GTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAGCC GGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCGCCCAGTGCGGTGCTGTCCCGCTGCT GCGCAGGCTCGGGGGCGAGCCCGGCCGGATCGACGACGCCGGCCTGCCGCAGCGGAGCACATCG TTGACCGATGCGGAGCGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCG CCAAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCGTCTTCCGCAAACTGG GGGTCAAGGGTCGCAAGCAGCTGCCGACCGCGCTGGCCGACGTGGAACAGACCTGA
SEQ ID NO: 51
ATGCCTGCCGTGGAGAGCTATGAACTGGACGCCCGCGATGACGAGCTCAGAAGACTGGAGGAGGC GGTAGGCCAGGCGGGCAACGGCCGGGGTGTGGTGGTCACCATCACCGGGCCGATCGCCTGCGGC AAGACCGAACTGCTCGACGCGGCCGCCGCGAAGAGCGACGCCATCACATTACGTGCGGTCTGCTC CGAGGAGGAACGGGCCCTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGGCGGTCGCCT CCCAGCTGCCGGATCCGGTCTCCATGGCCCTCCCGGGCGAGCACCTGTCGCCGGAGGCCGAGAAC CGGCTGCGCGGCGACCTCACCCGTACCCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCG GCATCGACGACATGCACCACGCCGACACCGCCTCTTTGAACTGCCTGCTCCACCTGGCCCGGAGG GTCGGCCCGGCCCGGATCGCCATGGTCCTCACCGAGCTGCGCCGGCTCACCCCGGCCCACTCCCA GTTCCACGCCGAGCTGCTCAGCCTGGGGCACCACCGCGAGATCGCGCTGCGCCCGCTCGGCCCGA AGCACATCGCCGAGCTGGCCCGCGCCGGCCTCGGTCCCGATGTCGACGAGGACGTGCTCACGGG GTTGTACCGGGCGACCGGCGGCAACCTGAACCTCGGCCACGGACTGATCAAGGATGTGCGGGAGG CCTGGGCGACGGGCGGGACGGGCATCAACGCGGGCCGCGCGTACCGGCTGGCGTACCTCGGTTC CCTCTACCGCTGCGGCCCGGTCCCGTTGCGGGTCGCACGGGTGGCCGCCGTGCTGGGCCAGAGC GCCAACACCACCCTGGTGCGCTGGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCGACCG AGATCCTCACCGAGGGCGGCCTGCTGCACGACCTGCGGTTCCCGCATCCGGCGGCCCGTTCGGTC GTACTCAACGACCTGTCCGCCCGGGAACGCCGCCGACTGCACCGGTCCGCTCTGGAAGTGCTGGA TGACGTACCCGTTGAAGTGGTCGCGCACCACCAGGCCGGTGCCGGTTTCATCCACGGTCCCAAGG CCGCCGAGATCTTCGCCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGCTGGACGCCGCGTC CGACTATCTGCAACTGGCCCACCACGCCTCCGACGACGCCGTCACCCGGGCCGCGCTGCGGGTCG AGGCCGTGGCGATCGAGCGCCGCCGCAACCCGCTGGCCTCCAGCCGCCACCTCGACGAGCTGAC CGTCGCCGCCCGTGCCGGTCTGCTCTCCCTCGAGCACGCCGCGCTGATGATCCGCTGGCTGGCTC TCGGCGGGCGGTCCGGCGAGGCGGCCGAGGTGCTGGCCGCGCAGCGCCCGCGTGCGGTCACCG ACCAGGACAGGGCCCACCTGCGGGCCGCCGAGGTATCGCTGGCGCTGGTCAGCCCGGGCGCGTC CGGCGTCAGCCCGGGTGCGTCCGGCCCGGATCGGCGGCCGCGTCCGCTCCCGCCGGATGAGCTC GCGAACCTGCCGAAGGCGGCCCGGCTTTGTGCGATCGCCGACAACGCCGTCATATCGGCCCTGCA CGGTCGTCCCGAGCTTGCCTCGGCCGAGGCGGAGAACGTCCTGAAGCAGGCTGACTCGGCGGCG GACGGCGCCACCGCCCTCTCCGCGCTGACGGCCTTGCTGTACGCGGAGAACACCGACACCGCTCA GCTCTGGGCCGACAAGCTCGTCTCCGAGACCGGGGCGTCGAACGAGGAGGAAGGCGCGGGCTAC GCGGGGCCGCGCGCCGAGACCGCGTTGCGCCGCGGCGACCTGGCCGCGGCGGTCGAGGCGGGC AGCGCCATTCTGGACCACCGGCGGGGGTCGTTGCTCGGCATCACCGCCGCGCTACCGCTGAGCAG CGCGGTAGCCGCCGCCATCCGGCTGGGCGAGACCGAGCGGGCGGAGAAGTGGCTCGCCGAGCCG CTGCCGGAGGCCATTCGGGACAGCCTGTTCGGGCTGCACCTGCTCTCGGCGCGCGGCCAGTACTG CCTCGCGACGGGCCGGCACGAGTCGGCGTACACGGCGTTCCGCACCTGCGGGGAACGGATGCGG AACTGGGGCGTCGACGTGCCGGGTCTGTCCCTGTGGCGCGTCGACGCCGCCGAGGCGCTGCTGC ACGGCCGCGACCGGGACGAGGGCCGACGGCTCATCGACGAGCAGCTCACCCATGCGATGGGACC CCGTTCCCGCGCTTTGACGCTGCGGGTGCAGGCGGCGTACAGCCCGCAGGCGCAGCGGGTCGAC CTGCTCGAAGAGGCGGCCGACCTGCTGCTCTCCTGCAACGACCAGTACGAGCGGGCGCGGGTGCT CGCCGATCTGAGCGAGGCGTTCAGCGCGCTCAGGCACCACAGCCGGGCGCGGGGACTGCTCCGG CAGGCCCGGCACCTGGCCGCCCAGTGCGGCGCGACCCCGCTGCTGCGCCGGCTCGGGGCCAAGC CCGGAGGCCCCGGCTGGCTGGAGGAATCCGGCCTGCCGCAGCGGATCAAGTCGCTGACCGACGC GGAGCGGCGGGTGGCGTCGCTGGCCGCCGGCGGCCAGACCAACCGCGTGATCGCCGACCAGCTC TTCGTCACGGCCAGCACGGTGGAGCAGCACCTCACGAACGTCTTCCGCAAGCTGGGCGTCAAGGG CCGCCAGCACCTGCCGGCCGAACTCGCCAACGCGGAATAG SEQ ID NO: 52
ATGCCTGCCGTGGAGAGCTATGAACTGGACGCCCGCGATGACGAGCTCAGAAGACTGGAGGAGGC GGTAGGCCAGGCGGGCAACGGCCGGGGTGTGGTGGTCACCATCACCGGGCCGATCGCCTGCGGC AAGACCGAACTGCTCGACGCGGCCGCCGCGAAGAGCGACGCCATCACACTGCGTGCGGTCTGCTC CGAGGAGGAACGGGCCCTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGGCGGTCGCCT CCCAGCTGCCGGATCCGGTCTCCATGGCCCTCCCGGGCGAGCACCTGTCGCCGGAGGCCGAGAAC CGGCTGCGCGGCGACCTCACCCGTACCCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCG GCATCGACGACATGCACCACGCCGACACCGCCTCTTTGAACTGCCTGCTCCACCTGGCCCGGAGG GTCGGCCCGGCCCGGATCGCCATGGTCCTCACCGAGCTGCGCCGGCTCACCCCGGCCCACTCCCA GTTCCACGCCGAGCTGCTCAGCCTGGGGCACCACCGCGAGATCGCGCTGCGCCCGCTCGGCCCGA AGCACATCGCCGAGCTGGCCCGCGCCGGCCTCGGTCCCGATGTCGACGAGGACGTGCTCACGGG GTTGTACCGGGCGACCGGCGGCAACCTGAACCTCGGCCACGGACTGATCAAGGATGTGCGGGAGG CCTGGGCGACGGGCGGGACGGGCATCAACGCGGGCCGCGCGTACCGGCTGGCGTACCTCGGTTC CCTCTACCGCTGCGGCCCGGTCCCGTTGCGGGTCGCACGGGTGGCCGCCGTGCTGGGCCAGAGC GCCAACACCACCCTGGTGCGCTGGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCGACCG AGATCCTCACCGAGGGCGGCCTGCTGCACGACCTGCGGTTCCCGCATCCGGCGGCCCGTTCGGTC GTACTCAACGACCTGTCCGCCCGGGAACGCCGCCGACTGCACCGGTCCGCTCTGGAAGTGCTGGA TGACGTACCCGTTGAAGTGGTCGCGCACCACCAGGCCGGTGCCGGTTTCATCCACGGTCCCAAGG CCGCCGAGATCTTCGCCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGCTGGACGCCGCGTC CGACTATCTGCAACTGGCCCACCACGCCTCCGACGACGCCGTCACCCGGGCCGCGCTGCGGGTCG AGGCCGTGGCGATCGAGCGCCGCCGCAACCCGCTGGCCTCCAGCCGCCACCTCGACGAGCTGAC CGTCGCCGCCCGTGCCGGTCTGCTCTCCCTCGAGCACGCCGCGCTGATGATCCGCTGGCTGGCTC TCGGCGGGCGGTCCGGCGAGGCGGCCGAGGTGCTGGCCGCGCAGCGCCCGCGTGCGGTCACCG ACCAGGACAGGGCCCACCTGCGGGCCGCCGAGGTATCGCTGGCGCTGGTCAGCCCGGGCGCGTC CGGCGTCAGCCCGGGTGCGTCCGGCCCGGATCGGCGGCCGCGTCCGCTCCCGCCGGATGAGCTC GCGAACCTGCCGAAGGCGGCCCGGCTTTGTGCGATCGCCGACAACGCCGTCATATCGGCCCTGCA CGGTCGTCCCGAGCTTGCCTCGGCCGAGGCGGAGAACGTCCTGAAGCAGGCTGACTCGGCGGCG GACGGCGCCACCGCCCTCTCCGCGCTGACGGCCTTGCTGTACGCGGAGAACACCGACACCGCTCA GCTCTGGGCCGACAAGCTCGTCTCCGAGACCGGGGCGTCGAACGAGGAGGAAGGCGCGGGCTAC GCGGGGCCGCGCGCCGAGACCGCGTTGCGCCGCGGCGACCTGGCCGCGGCGGTCGAGGCGGGC AGCGCCATTCTGGACCACCGGCGGGGGTCGTTGCTCGGCATCACCGCCGCGCTACCGCTGAGCAG CGCGGTAGCCGCCGCCATCCGGCTGGGCGAGACCGAGCGGGCGGAGAAGTGGCTCGCCGAGCCG CTGCCGGAGGCCATTCGGGACAGCCTGTTCGGGCTGCACCTGCTCTCGGCGCGCGGCCAGTACTG CCTCGCGACGGGCCGGCACGAGTCGGCGTACACGGCGTTCCGCACCTGCGGGGAACGGATGCGG AACTGGGGCGTCGACGTGCCGGGTCTGTCCCTGTGGCGCGTCGACGCCGCCGAGGCGCTGCTGC ACGGCCGCGACCGGGACGAGGGCCGACGGCTCATCGACGAGCAGCTCACCCATGCGATGGGACC CCGTTCCCGCGCTTTGACGCTGCGGGTGCAGGCGGCGTACAGCCCGCAGGCGCAGCGGGTCGAC CTGCTCGAAGAGGCGGCCGACCTGCTGCTCTCCTGCAACGACCAGTACGAGCGGGCGCGGGTGCT CGCCGATCTGAGCGAGGCGTTCAGCGCGCTCAGGCACCACAGCCGGGCGCGGGGACTGCTCCGG CAGGCCCGGCACCTGGCCGCCCAGTGCGGCGCGACCCCGCTGCTGCGCCGGCTCGGGGCCAAGC CCGGAGGCCCCGGCTGGCTGGAGGAATCCGGCCTGCCGCAGCGGATCAAGTCGCTGACCGACGC GGAGCGGCGGGTGGCGTCGCTGGCCGCCGGCGGCCAGACCAACCGCGTGATCGCCGACCAGCTC TTCGTCACGGCCAGCACGGTGGAGCAGCACCTCACGAACGTCTTCCGCAAGCTGGGCGTCAAGGG CCGCCAGCACCTGCCGGCCGAACTCGCCAACGCGGAATAG
SEQ ID NO: 53
GTGAAGCGCAACGATCTGGTTGCCCGCGATGGCGAGCTCAGGTGGATGCAAGAGATTCTCAGTCAG GCGAGCGAGGGCCGGGGGGCCGTGGTCACCATCACGGGGGCGATCGCCTGTGGCAAGACGGTGC TGCTGGACGCCGCGGCAGCCAGTCAAGACGTGATCCAACTGCGTGCGGTCTGCTCGGCGGAGGAG CAGGAGCTGCCGTACGCGATGGTCGGACAACTACTCGACAATCCGGTGCTCGCCGCGCGAGTGCC GGCCCTGGGCAACCTGGCTGCGGCGGGCGAGCGGCTGCTGCCGGGCACCGAGAACAGGATCCGG CGGGAGCTCACCCGCACCCTGCTGGCTCTCGCCGACGAACGACCGGTGCTGATCGGCGTCGACGA CATGCACCATGCGGACCCCGCCTCGCTGGACTGCCTGCTGCACCTGGCCCGGCGGGTCGGCCCG GCCCGCATCGCGATCGTTCTGACCGAGTTGCGCCGGCTCACCCCGGCTCACTCGCGCTTCCAGTCC GAGCTGCTCAGCCTGCGGTACCACCACGAGATCGGGTTGCAGCCGCTCACCGCGGAGCACACCGC CGACCTGGCCCGCGTCGGCCTCGGTGCCGAGGTCGACGACGACGTGCTCACCGAGCTCTACGAGG CGACCGGCGGCAACCCGAGTCTGTGCTGCGGCCTGATCAGGGACGTGCGGCAGGACTGGGAGGC CGGGGTCACCGGTATCCACGTCGGCCGGGCGTACCGGCTGGCCTATCTCAGTTCGCTCTACCGCT GCGGCCCGGCGGCGCTGCGGACCGCCCGCGCGGCCGCGGTGCTGGGCGACAGCGCCGACGCCT GCCTGATCCGCCGGGTCAGCGGCCTCGGTACGGAGGCCGTGGGCCAGGCGATCCAGCAGCTCAC CGAGGGCGGCCTGCTGCGTGACCAGCAGTTCCCGCACCCGGCGGCCCGCTCGGTCGTGCTCGAC GACATGTCCGCGCAGGAACGCCACGCGATGTATCGCAGCGCCCGGGAGGCAGCCGCCGAAGGTCA GGCCGACCCCGGCACCCCGGGCGAGCCGCGGGCGGCTACGGCGTACGCCGGGTGTGGTGAGCAA GCCGGTGACTACCCGGAGCCGGCCGGCCGGGCCTGCGTGGACGGTGCCGGTCCGGCCGAGTACT GCGGCGACCCGCACGGCGCCGACGACGACCCGGACGAGCTGGTCGCCGCGCTGGGCGGGCTGCT GCCGAGCCGGCTCGTGGCGATGAAGATCCGGCGCCTGGCGGTGGCCGGGCGCCCCGGGGCGGC TGCCGAGCTGCTGACCTCGCAGCGGTTGCACGCGGTGACCAGCGAGGACCGGGCCAGCCTGCGG GCCGCCGAGGTGGCGCTCGCCACGCTGTGGCCGGGTGCGACCGGCCCGGACCGGCATCCGCTCA CGGAGCAGGAGGCGGCGAGCCTGCCGGAGGGTCCGCGCCTGCTCGCTGCCGCCGACGATGCCGT CGGGGCCGCCCTGCGCGGTCGCGCCGAGTACGCCGCGGCCGAGGCGGAGAACGTCCTGCGGCAC GCCGATCCGGCAGCCGGTGGTGACGCCTACGCCGCCATGATCGCCCTGCTGTACACGGAGCACCC CGAGAACGTGCTGTTCTGGGCCGACAAGCTCGACGCGGGCCGCCCCGACGAGGAGACCAGTTATC CCGGGCTGCGGGCCGAGACCGCGGTGCGGCTCGGTGACCTGGAAACGGCGATGGAGCTGGGCCG CACGGTGCTGGACCAGCGGCGGCTGCCGTCCCTGGGTGTCGCCGCGGGCCTGCTCCTGGGCGGC GCGGTGACGGCCGCCATCCGGCTCGGCGACCTCGACCGGGCGGAGAAGTGGCTCGCCGAGCCGA TCCCCGACGCCATCCGTACCAGCCTCTACGGCCTGCACGTGCTGGCCGCGCGGGGCCGGCTCGAC CTGGCCGCGGGCCGCTACGAGGCGGCGTACACGGCGTTCCGGCTGTGTGGCGAGCGGATGGCAG GCTGGGATGCCGATGTCTCCGGGCTGGCGCTGTGGCGCGTCGACGCCGCCGAGGCCCTGCTGTC CGCGGGCATCCGCCCGGACGAGGGCCGCAAGCTCATCGACGACCAGCTCACCCGTGAGATGGGG GCCCGCTCCCGGGCGCTGACGCTGCGGGCGCAAGCGGCGTACAGCCTGCCGGTGCACCGGGTGG GCCTGCTCGACGAGGCGGCCGGCCTGCTGCTCGCCTGCCATGACGGGTACGAGCGGGCGCGGGT GCTCGCGGACCTGGGGGAGACCCTGCGCACGCTGCGGCACACCGACGCGGCCCAGCGGGTGCTC CGGCAGGCCGAGCAGGCGGCCGCGCGGTGCGGGTCGGTCCCGCTGCTGCGGCGGCTCGGGGCC GAACCCGTACGCATCGGCACCCGGCGTGGTGAACCCGGCCTGCCGCAGCGGATCAGGCTGCTGAC CGATGCCGAGCGGCGGGTTGCCGCGATGGCCGCCGCCGGGCAGACCAACCGGGAGATCGCCGGT CGGCTCTTCGTCACGGCCAGCACGGTGGAGCAGCACCTGACCAGCGTCTTCCGCAAGCTGGGCGT CAAGGGCCGCCGGTTCCTGCCGACCGAGCTCGCCCAAGCCGTCTGA
SEQ ID NO: 54
ATGCCTGCCGTGAAGCGCAACGATCTGGTTGCCCGCGATGGCGAGCTCAGGTGGATGCAAGAGATT CTCAGTCAGGCGAGCGAGGGCCGGGGGGCCGTGGTCACCATCACGGGGGCGATCGCCTGTGGCA AGACGGTGCTGCTGGACGCCGCGGCAGCCAGTCAAGACGTGATCCAACTGCGTGCGGTCTGCTCG GCGGAGGAGCAGGAGCTGCCGTACGCGATGGTCGGACAACTACTCGACAATCCGGTGCTCGCCGC GCGAGTGCCGGCCCTGGGCAACCTGGCTGCGGCGGGCGAGCGGCTGCTGCCGGGCACCGAGAAC AGGATCCGGCGGGAGCTCACCCGCACCCTGCTGGCTCTCGCCGACGAACGACCGGTGCTGATCGG CGTCGACGACATGCACCATGCGGACCCCGCCTCGCTGGACTGCCTGCTGCACCTGGCCCGGCGGG TCGGCCCGGCCCGCATCGCGATCGTTCTGACCGAGTTGCGCCGGCTCACCCCGGCTCACTCGCGC TTCCAGTCCGAGCTGCTCAGCCTGCGGTACCACCACGAGATCGGGTTGCAGCCGCTCACCGCGGA GCACACCGCCGACCTGGCCCGCGTCGGCCTCGGTGCCGAGGTCGACGACGACGTGCTCACCGAG CTCTACGAGGCGACCGGCGGCAACCCGAGTCTGTGCTGCGGCCTGATCAGGGACGTGCGGCAGGA CTGGGAGGCCGGGGTCACCGGTATCCACGTCGGCCGGGCGTACCGGCTGGCCTATCTCAGTTCGC TCTACCGCTGCGGCCCGGCGGCGCTGCGGACCGCCCGCGCGGCCGCGGTGCTGGGCGACAGCG CCGACGCCTGCCTGATCCGCCGGGTCAGCGGCCTCGGTACGGAGGCCGTGGGCCAGGCGATCCA GCAGCTCACCGAGGGCGGCCTGCTGCGTGACCAGCAGTTCCCGCACCCGGCGGCCCGCTCGGTC GTGCTCGACGACATGTCCGCGCAGGAACGCCACGCGATGTATCGCAGCGCCCGGGAGGCAGCCGC CGAAGGTCAGGCCGACCCCGGCACCCCGGGCGAGCCGCGGGCGGCTACGGCGTACGCCGGGTGT GGTGAGCAAGCCGGTGACTACCCGGAGCCGGCCGGCCGGGCCTGCGTGGACGGTGCCGGTCCGG CCGAGTACTGCGGCGACCCGCACGGCGCCGACGACGACCCGGACGAGCTGGTCGCCGCGCTGGG CGGGCTGCTGCCGAGCCGGCTCGTGGCGATGAAGATCCGGCGCCTGGCGGTGGCCGGGCGCCCC GGGGCGGCTGCCGAGCTGCTGACCTCGCAGCGGTTGCACGCGGTGACCAGCGAGGACCGGGCCA GCCTGCGGGCCGCCGAGGTGGCGCTCGCCACGCTGTGGCCGGGTGCGACCGGCCCGGACCGGC ATCCGCTCACGGAGCAGGAGGCGGCGAGCCTGCCGGAGGGTCCGCGCCTGCTCGCTGCCGCCGA CGATGCCGTCGGGGCCGCCCTGCGCGGTCGCGCCGAGTACGCCGCGGCCGAGGCGGAGAACGTC CTGCGGCACGCCGATCCGGCAGCCGGTGGTGACGCCTACGCCGCCATGATCGCCCTGCTGTACAC GGAGCACCCCGAGAACGTGCTGTTCTGGGCCGACAAGCTCGACGCGGGCCGCCCCGACGAGGAG ACCAGTTATCCCGGGCTGCGGGCCGAGACCGCGGTGCGGCTCGGTGACCTGGAAACGGCGATGGA GCTGGGCCGCACGGTGCTGGACCAGCGGCGGCTGCCGTCCCTGGGTGTCGCCGCGGGCCTGCTC CTGGGCGGCGCGGTGACGGCCGCCATCCGGCTCGGCGACCTCGACCGGGCGGAGAAGTGGCTCG CCGAGCCGATCCCCGACGCCATCCGTACCAGCCTCTACGGCCTGCACGTGCTGGCCGCGCGGGGC CGGCTCGACCTGGCCGCGGGCCGCTACGAGGCGGCGTACACGGCGTTCCGGCTGTGTGGCGAGC GGATGGCAGGCTGGGATGCCGATGTCTCCGGGCTGGCGCTGTGGCGCGTCGACGCCGCCGAGGC CCTGCTGTCCGCGGGCATCCGCCCGGACGAGGGCCGCAAGCTCATCGACGACCAGCTCACCCGTG AGATGGGGGCCCGCTCCCGGGCGCTGACGCTGCGGGCGCAAGCGGCGTACAGCCTGCCGGTGCA CCGGGTGGGCCTGCTCGACGAGGCGGCCGGCCTGCTGCTCGCCTGCCATGACGGGTACGAGCGG GCGCGGGTGCTCGCGGACCTGGGGGAGACCCTGCGCACGCTGCGGCACACCGACGCGGCCCAGC GGGTGCTCCGGCAGGCCGAGCAGGCGGCCGCGCGGTGCGGGTCGGTCCCGCTGCTGCGGCGGC TCGGGGCCGAACCCGTACGCATCGGCACCCGGCGTGGTGAACCCGGCCTGCCGCAGCGGATCAG GCTGCTGACCGATGCCGAGCGGCGGGTTGCCGCGATGGCCGCCGCCGGGCAGACCAACCGGGAG ATCGCCGGTCGGCTCTTCGTCACGGCCAGCACGGTGGAGCAGCACCTGACCAGCGTCTTCCGCAA GCTGGGCGTCAAGGGCCGCCGGTTCCTGCCGACCGAGCTCGCCCAAGCCGTCTGA
SEQ ID NO: 55
GTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAA GGCTGAGGCCATCATTCTGCGCGCGGTCTGCGCGCCAGAAGAGCGGGCTATGCCGTACGCCATGA TCGGGCAGCTCATCGACGACCCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGC CCAGGGCGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTG CTGGCGCTTGCCGTGGACCGGCCTGTGCTGATCGGCGTCGACGATGTGCATCACGCCGACACCGC CTCTTTGAACTGTCTGCTGCATTTGGCCCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACC GAGTTGCGCAGCCTCACCCCTACTCAGTCACGGTTCAAGGCGGAGCTGCTCAGCCTGCCATACCAC CACGAGATCGCGCTGCGTCCATTCGGACCGGAGCAATCGGCGGAGCTGGCTCGCGCCGCCTTCGG CCCGGGCCTCGCCGAGGATGTGCTCGCGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCA GCCGTGGACTGATCAGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCTTTCGAGGCGGG CCGCGCGTTCCGGCTGGCGTACCTCAGCTCGCTCTACCGCTGTGGCCCGGTCGCGCTGCGGGTCG CCCGAGTGGCTGCCGTGCTGGGCCCAAGCGCCACCACCACGCTGGTGCGCCGGCTAAGCGGGCT CAGCGCGGAGACGATAGACCGGGCAACCAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCAGC AGTTCCCGCACCCGGCCGCCCGCTCGGTGGTGCTCGATGACATGTCCGCCCAGGAACGACGCAGC CTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGT CGGCGCCGGTCTCATACACGGGCCCAAGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGG TCGTACGCAACGAGTTGGGCGACGCGGCCGAATACCTGCAACTGGCTCACCGGGCCTCCGACGAT GTCTCCACCCGGGCCGCCTTACGGGTCGAGGCCGTGGCCATCGAGCGCCGCCGCAATCCGCTGGC CTCCAGTCGGCACATGGACGAACTGAGCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATG CGGCGCTGGCCGTCTTCTGGCTAGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAAGTGCTGGC GTCGGAACGCCCGCTCGCGACCACCGATCAGAACCGGGCCCACCTGCGATTTGTCGAGGTGACTC TCGCGCTGTTCTCTCCCGGCGCCTTCGGATCGGACCGGCGCCCACCTCCGCTGACGCCGGACGAA CTCGCCAGCCTGCCGAAGGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTT GCACGGCCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAG CCGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTACGCGGAGAACACCGAGTCCGCTCATATCT GGGCCGACAAGCTGGGCAGCACGAATGCCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGG CCCGTGCGCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCGCC GTCCTGGACGACCGGTCGCTGCCGTCGCTCGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGAC GGCCGCCGCTGTCCGGCTGGGCGAACTCGAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCG AACGGCGTCCAGGACAGCCTTTTCGGTCTGCACCTGCTCTCGGCGTACGGCCAGTACAGCCTCGCG ATGGGCCGATATGAATCAGCTCACCGGGCGTTTCGCACCTGCGGAGAACGTATGCGCAGCTGGGAT GTTGACGTGCCTGGTCTGGCCCTGTGGCGTGTCGACGCCGCCGAGGCGCTGCTCAGCCTCGACCG GAACGAGGGCCAGCGGCTCATCGACGAACAACTCACCCGTCCGATGGGGCCTCGTTCCCACGCGT TAACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCG GCCGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGA CACGCTCAGCGCGCTCAGACGCTATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGG CCACCCAGTGCGGTGCTGTCCCGCTGCTGCGCAGGCTCGGGGGCGAGCCCGGCCGGATCGACGA CGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAGCGGCGGGTGGCGGCGCTGGCC GCGGCCGGACAGACCAACCGGGAGATCGCCGAACAGCTGTTCGTCACGGCCAGCACAGTGGAACA GCACCTCACAAGCGTCTTCCGCAAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTGG CCGACGTGGAACAGACCTGA
SEQ ID NO: 56
ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGACGAACTCGGTATTCTA CAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGGGCGTCGTGGTCACCGTCACCGGCCCAATCGC CTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGG TCTGCGCGCCAGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGACCCGGCG CTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCCCAGGGCGGGCAGCTGTCGCTGAGGG CCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTGCTGGCGCTTGCCGTGGACCGGCCTGTG CTGATCGGCGTCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATTTGGCC CGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCAGCCTCACCCCTACTCAG TCACGGTTCAAGGCGGAGCTGCTCAGCCTGCCATACCACCACGAGATCGCGCTGCGTCCATTCGGA CCGGAGCAATCGGCGGAGCTGGCTCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCTCG CGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGACTGATCAGCGATGTGCGGG AGGCCCTGGCCAACGGAGAGAGCGCTTTCGAGGCGGGCCGCGCGTTCCGGCTGGCGTACCTCAG CTCGCTCTACCGCTGTGGCCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAA GCGCCACCACCACGCTGGTGCGCCGGCTAAGCGGGCTCAGCGCGGAGACGATAGACCGGGCAAC CAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCAGCAGTTCCCGCACCCGGCCGCCCGCTCGG TGGTGCTCGATGACATGTCCGCCCAGGAACGACGCAGCCTGCACACTCTCGCCCTGGAACTGCTGG ACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCAAG GCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTACGCAACGAGTTGGGCGACGCGGC CGAATACCTGCAACTGGCTCACCGGGCCTCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGTCG AGGCCGTGGCCATCGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAACTGAGC GCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCCGTCTTCTGGCTAGCCGA CGGCGGGCGATCCGGCGAGGCAGCCGAAGTGCTGGCGTCGGAACGCCCGCTCGCGACCACCGAT CAGAACCGGGCCCACCTGCGATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGGA TCGGACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAAGGCGGCCTGGC AATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGCACGGCCATCCAGAACTTGCCACCGCT CAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGC CCTGTTGTACGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCACGAATGC CGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGAGATCGCCCTGCGGCGC GGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCGCCGTCCTGGACGACCGGTCGCTGCCGTCGCT CGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTCG AGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGACAGCCTTTTCGGTCTG CACCTGCTCTCGGCGTACGGCCAGTACAGCCTCGCGATGGGCCGATATGAATCAGCTCACCGGGC GTTTCGCACCTGCGGAGAACGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGC GTGTCGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATCGACGAA CAACTCACCCGTCCGATGGGGCCTCGTTCCCACGCGCTGACGCTGCGGATCAAGGCGGCATACCT CCCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACC CGTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAGC CGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCAGTGCGGTGCTGTCCCGCTGCT GCGCAGGCTCGGGGGCGAGCCCGGCCGGATCGACGACGCCGGCCTGCCGCAGCGGAGCACATCG TTGACCGATGCGGAGCGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCG CCGAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCGTCTTCCGCAAGCTGG GCGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTGGCCGACGTGGAACAGACCTGA SEQ ID NO: 57
GTGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCCCGCGAGGACGAACTCGGCATTCTG CAGAGGTCTCTGGAAGAAGCAGGCAGCGGCCAGGGCGCCGTGGTCACCGTCACCGGCCCGATCG CCTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAAGGCTGACGCCATCATTCTGCGCGCG GTCTGCGCGCCCGAAGAGCGCGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGACCCGGC GCTCGCGCATCGGGCGCCGGAGCTGGCTGATCGGATAGCCCAGGGCGGGCATCTGTCGCTGAGG GCCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTGCTGGCGCTTGCCGTCGACCGGCCTGT GCTGATCGGCGTCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATTTAGC CCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCAGCCTCACCCCTACTCA GTCACGATTCAAGGCGGAGCTGCTCAGCCTGCCGTACCACCACGAGATCGCGCTGCGTCCACTCG GACCGGAGCAATCGGCGGAGCTGGCCCACGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGCGGGGTTGTATGGGATGACCAGGGGCAACCTGAGTCTCAGCCGTGGACTGATCAGCGATGTGC GGGAGGCCCAGGCCAACGGAGAGAGCGCTTTCGAGGTGGGCCGCGCGTTCCGGCTGGCGTACCT CAGCTCGCTCTACCGCTGTGGCCCGATCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCC CAAGCGCCACCACCACGCTGGTGCGCCGTCTAAGCGGGCTCAGCGCGGAGACGATAGACCGGGCA ACCAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCACCAGTTCCCGCACCCGGCCGCCCGCTC GGTGGTGCTCGATGACATGTCCGCCCAGGAACGACGCAGCCTGCACACTCTCGCCCTGGAACTGCT GGACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCA AGGCTGCGGAGATATTCGCCAGGGCTGGCCAGGCTCTGGTTGTACGCAACGAGTTGGGCGACGCG GCCGAATACCTGCAACTGGCTCACCGAGCCTCCGACGATGTCTCCACCCGGGCCGCCTTACGGGTC GAGGCCGTGGCAATCGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGTCACATGGACGAGCTGAG CGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCAGCGCTGGCTGTCTTCTGGCTGGCCG ACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCTGGCGTCGGAACACCCGCTCGCGACCACCGA TCAGAACCGAGCACACCTGCGATTTGCCGAGGTGACTCTCGCGCTGTTCTGTCCCGGCGCCTTCGG GTCGGACCGGCGCCCACCTCCGCTGGCGCCGGACGAGCTCGCCAGCTTGCCGAAGGCGGCCTGG CAATGCGCGGTCGCCGACAACGCGGTCATGACAGCGTTGCATGCTCATCCAGAACTTGCCACCGCT CAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAGCCGACGCAATCCCCGCCGCACTGATCGC CCTGTTGTACGCAGAGAACACCGAGTCCGCTCAGATCTGGGCCGACAAGCTGGGCAGCACCAATGC CGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGAGATCGCCCTGCGGCGC GGCGACCTGGCCACGGCGTTCGAGGCTGGTGGCACCGTCCTGGACGACCGGCCGCTGCCGTCGC TCGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGACGGCAGCCGCTGTCCGCCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCTGAGCCGCTTCCGAACGGTGTCCAGGACAGCCTTTTCGGTCT GCACCTGCTCTCGGCGCACGGCCAGTACAGCCTCGCGATGGGCCGATATGAATCGGCTCACCGGG CGTTTCACACCTGCGGAGAACGTATGCGCAGCTGGGGTGTTGACGTGCCTGGTCTAGCCCTGTGGC GTGTCGACGCCGCCGAGGCACTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATCGACGAA CAACTCGCCCGTCCGATGGGACCTCGTTCCCGCGCATTAACGCTGCGGATCAAGGCGGCATACCTC CCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCAGCTGAGCTGCTGCTCTCCTGCCCCGACCC GTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAGCC GGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCAGTGCGGTGCTGTCCCGCTGCTG CGCCGACTCGGGGGCGAGCCCGGCCGGATCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGT TGACCGATGCGGAGCGGCGGGTGTCGGCCCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CAAACAGCTATTCGTCACGGCCAGCACCGTGGAACAGCACCTCACAAGCGTCTTCCGCAAGCTGGG CGTTAAGGGCCGCAGGCAGCTACCGACCGCGCTGGCCGACGTGGAATAG SEQ ID NO: 58
ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCCCGCGAGGACGAACTCGGCATTCTG CAGAGGTCTCTGGAAGAAGCAGGCAGCGGCCAGGGCGCCGTGGTCACCGTCACCGGCCCGATCG CCTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAAGGCTGACGCCATCATTCTGCGCGCG GTCTGCGCGCCCGAAGAGCGCGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGACCCGGC GCTCGCGCATCGGGCGCCGGAGCTGGCTGATCGGATAGCCCAGGGCGGGCATCTGTCGCTGAGG GCCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTGCTGGCGCTTGCCGTCGACCGGCCTGT GCTGATCGGCGTCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATCTGGC CCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCAGCCTCACCCCTACTCA GTCACGATTCAAGGCGGAGCTGCTCAGCCTGCCGTACCACCACGAGATCGCGCTGCGTCCACTCG GACCGGAGCAATCGGCGGAGCTGGCCCACGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGCGGGGTTGTATGGGATGACCAGGGGCAACCTGAGTCTCAGCCGTGGACTGATCAGCGATGTGC GGGAGGCCCAGGCCAACGGAGAGAGCGCTTTCGAGGTGGGCCGCGCGTTCCGGCTGGCGTACCT CAGCTCGCTCTACCGCTGTGGCCCGATCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCC CAAGCGCCACCACCACGCTGGTGCGCCGTCTAAGCGGGCTCAGCGCGGAGACGATAGACCGGGCA ACCAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCACCAGTTCCCGCACCCGGCCGCCCGCTC GGTGGTGCTCGATGACATGTCCGCCCAGGAACGACGCAGCCTGCACACTCTCGCCCTGGAACTGCT GGACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCA AGGCTGCGGAGATATTCGCCAGGGCTGGCCAGGCTCTGGTTGTACGCAACGAGTTGGGCGACGCG GCCGAATACCTGCAACTGGCTCACCGAGCCTCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGT CGAGGCCGTGGCAATCGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGTCACATGGACGAGCTGA GCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCAGCGCTGGCTGTCTTCTGGCTGGCC GACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCTGGCGTCGGAACACCCGCTCGCGACCACCG ATCAGAACCGAGCACACCTGCGATTTGCCGAGGTGACTCTCGCGCTGTTCTGTCCCGGCGCCTTCG GGTCGGACCGGCGCCCACCTCCGCTGGCGCCGGACGAGCTCGCCAGCTTGCCGAAGGCGGCCTG GCAATGCGCGGTCGCCGACAACGCGGTCATGACAGCGTTGCATGCTCATCCAGAACTTGCCACCGC TCAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAGCCGACGCAATCCCCGCCGCACTGATCG CCCTGTTGTACGCAGAGAACACCGAGTCCGCTCAGATCTGGGCCGACAAGCTGGGCAGCACCAATG CCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGAGATCGCCCTGCGGCG CGGCGACCTGGCCACGGCGTTCGAGGCTGGTGGCACCGTCCTGGACGACCGGCCGCTGCCGTCG CTCGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGACGGCAGCCGCTGTCCGCCTGGGCGAACT CGAGCGTGCGGAGAAGCTGCTCGCTGAGCCGCTTCCGAACGGTGTCCAGGACAGCCTTTTCGGTCT GCACCTGCTCTCGGCGCACGGCCAGTACAGCCTCGCGATGGGCCGATATGAATCGGCTCACCGGG CGTTTCACACCTGCGGAGAACGTATGCGCAGCTGGGGTGTTGACGTGCCTGGTCTAGCCCTGTGGC GTGTCGACGCCGCCGAGGCACTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATCGACGAA CAACTCGCCCGTCCGATGGGACCTCGTTCCCGCGCACTGACGCTGCGGATCAAGGCGGCATACCT CCCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCAGCTGAGCTGCTGCTCTCCTGCCCCGACC CGTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAGC CGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCAGTGCGGTGCTGTCCCGCTGCT GCGCCGACTCGGGGGCGAGCCCGGCCGGATCGACGACGCCGGCCTGCCGCAGCGGAGCACATCG TTGACCGATGCGGAGCGGCGGGTGTCGGCCCTGGCCGCGGCCGGACAGACCAACCGGGAGATCG CCAAACAGCTATTCGTCACGGCCAGCACCGTGGAACAGCACCTCACAAGCGTCTTCCGCAAGCTGG GCGTTAAGGGCCGCAGGCAGCTACCGACCGCGCTGGCCGACGTGGAATAG
SEQ ID NO: 59
GTGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGACGAACTCGGCATTCTA CAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGGGCGTCGTGGTCACCGTCACCGGCCCAATCGC CTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGG TCTGCGCGCCCGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGACCCGGCG CTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCCCAGGGCGGGCAGCTGTCGCTGAGGG CCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTGCTGGCGCTTGCCGTGCACCGGCCTGTG CTGATCGGCGTCGATGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATTTGGCG CGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCAGCCTCACCCCTACTCAG TCACGATTCAAGGCGGAGCTGCTCAGCCTGCCGTACCACCACGAGATCGCGCTGCGTCCATTCGGA CCGGAGCAATCGGCGGAGCTGGCTCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCTCG CGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGACTGATCAGCGATGTGCGGG AGGCCCTGGCCAACGGAGAGAGCGCTTTCGAGGCGGGCCGCGCGTTCCGGCTGGCGTACCTCAG CTCGCTCTACCGCTGTGGCCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAA GCGCCACCACCACGCTGGTGCGCCGGCTAAGCGGGCTCAGCGCGGAGACGATAGACCGGGCAAC CAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGCAGTTTCCGCACCCGGCCGCCCGCTCGG TGGTGCTCGATGACATGTCCGCCCAGGAACGACGCGGCCTGCACACTCTCGCCCTGGAACTGCTG GACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCAA GGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTACGCAACGAGTTGGGCGACGCGG CCGAATACCTGCAACTGGCTCACCGGGCCTCCGACGATGTCTCCACCCGGGCCGCCTTACGGGTC GAGGCCGTGGCGATCGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAGCTGAG CGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCCGTCTTCTGGCTGGCCG ACGGCGGGCGATCCGGCGAGGCAGCCCAGGTGCTGGCGTCGGAACGCCCGCTCGCGACCACCGA TCAGAACCGGGCCCACCTGCGATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGG ATCGGACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAAGGCGGCCTGG CAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGCACGGCCATCCAGAACTTGCCACCGC TCAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCG CCCTGTTGTACGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCATGAATG CCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGAGATCGCCCTGCGGCG CGGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCACCGTCCTGGACGACCGGTCACTGCCGTCGC TCGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGACAGCCTTTTCGGTCT GCACCTGCTCTCGGCGTACGGCCAGTACAGCCTCGCGATGGGCCGATATGAATCGGCTCACCGGG CGTTTCGCACCTGCGGAGAACGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGC GTGTCGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATCGACGAA CAACTCACCCGTCCGATGGGACCTCGTTCCCGCGCGTTAACGCTGCGGATCAAGGCGGCATACCTC CCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCC GTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAGCC GGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCAGTGCGGTGCTGTCCCGCTGCTG CGCCGACTCGGGGGCGAGCCCGGCCGGATCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGT TGACCGATGCGGAGCGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CGAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCGTCTTCCGCAAGCTGGG CGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTGGCCGACGTGGAACAGACCTGA
SEQ ID NO: 60
ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGACGAACTCGGCATTCTA CAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGGGCGTCGTGGTCACCGTCACCGGCCCAATCGC CTGCGGCAAGACAGAACTGCTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGG TCTGCGCGCCCGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGACCCGGCG CTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCCCAGGGCGGGCAGCTGTCGCTGAGGG CCGAGAACCGACTGCGCAGGGATCTCACCCGTGCCCTGCTGGCGCTTGCCGTGCACCGGCCTGTG CTGATCGGCGTCGATGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATTTGGCG CGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCAGCCTCACCCCTACTCAG TCACGATTCAAGGCGGAGCTGCTCAGCCTGCCGTACCACCACGAGATCGCGCTGCGTCCATTCGGA CCGGAGCAATCGGCGGAGCTGGCTCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCTCG CGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGACTGATCAGCGATGTGCGGG AGGCCCTGGCCAACGGAGAGAGCGCTTTCGAGGCGGGCCGCGCGTTCCGGCTGGCGTACCTCAG CTCGCTCTACCGCTGTGGCCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAA GCGCCACCACCACGCTGGTGCGCCGGCTAAGCGGGCTCAGCGCGGAGACGATAGACCGGGCAAC CAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGCAGTTTCCGCACCCGGCCGCCCGCTCGG TGGTGCTCGATGACATGTCCGCCCAGGAACGACGCGGCCTGCACACTCTCGCCCTGGAACTGCTG GACGAGGCGCCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCAA GGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTACGCAACGAGTTGGGCGACGCGG CCGAATACCTGCAACTGGCTCACCGGGCCTCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGTC GAGGCCGTGGCGATCGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAGCTGAG CGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCCGTCTTCTGGCTGGCCG ACGGCGGGCGATCCGGCGAGGCAGCCCAGGTGCTGGCGTCGGAACGCCCGCTCGCGACCACCGA TCAGAACCGGGCCCACCTGCGATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGG ATCGGACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAAGGCGGCCTGG CAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGCACGGCCATCCAGAACTTGCCACCGC TCAGGCGGAAACAGTTCTGCGGCAGGCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCG CCCTGTTGTACGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCATGAATG CCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGAGATCGCCCTGCGGCG CGGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCACCGTCCTGGACGACCGGTCACTGCCGTCGC TCGGCATCACCGCCGCATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGACAGCCTTTTCGGTCT GCACCTGCTCTCGGCGTACGGCCAGTACAGCCTCGCGATGGGCCGATATGAATCGGCTCACCGGG CGTTTCGCACCTGCGGAGAACGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGC GTGTCGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATCGACGAA CAACTCACCCGTCCGATGGGACCTCGTTCCCGCGCGCTGACGCTGCGGATCAAGGCGGCATACCT CCCGCGGACGAAGCGGATCCCCCTGCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACC CGTACGAGCAAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAGC CGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCAGTGCGGTGCTGTCCCGCTGCT GCGCCGACTCGGGGGCGAGCCCGGCCGGATCGACGACGCCGGCCTGCCGCAGCGGAGCACATCG TTGACCGATGCGGAGCGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCG CCGAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCGTCTTCCGCAAGCTGG GCGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTGGCCGACGTGGAACAGACCTGA
SEQ ID NO: 61
GTGCGAGCTATTAATGCGTCCGACACCGGTCCTGAACTGGTCGCCCGCGAAGACGAACTGGGACGT GTACGAAGTGCCCTGAACCGAGCGAACGGCGGCCAAGGTGTCCTGATCTCCATTACCGGTCCGATC GCCTGCGGCAAGACCGAACTGCTTGAGGCTGCCGCCTCGGAAGTTGACGCCATCACTCTGCGCGC GGTCTGTGCCGCCGAGGAACGGGCGATACCTTATGCCCTGATCGGGCAGCTTATCGACAACCCCGC GCTCGGCATTCCGGTTCCGGATCCGGCCGGCCTGACCGCCCAGGGCGGACGACTGTCATCGAGCG CCGAGAACCGACTGCGTCGCGACCTCACCCGTGCCCTGCTGACGCTCGCCACCGACCGGCTGGTG CTGATCTGTGTCGATGACGTGCAGCACGCCGACAACGCCTCGTTGAGCTGCCTTCTGTATCTGGCC CGACGGCTTGTCCCGGCTCGAATCGCTCTGGTATTCACCGAGTTGCGAGTCCTCACCTCGTCTCAG TTACGGTTCAACGCGGAGCTGCTCAGCTTGCGGAACCACTGCGAGATCGCGCTGCGCCCACTCGG CCCGGGGCATGCGGCCGAGCTGGCCCGCGCCACCCTCGGCCCCGGCCTCTCCGACGAAACACTC ACGGAGCTGTACCGGGTGACCGGAGGCAACCTGAGTCTCAGCCGCGGGCTGATCGACGATGTGCG GGACGCCTGGGCACGAGGGGAAACGGGCGTCCAGGTGGGCCGGGCGTTCCGGCTGGCCTACCTC GGTTCCCTCCACCGCTGTGGTCCGCTGGCGTTGCGGGTCGCCCGCGTAGCCGCCGTACTGGGCCC GAGCGCCACCAGCGTCCTGGTGCGCCGGATCAGTGGGCTCAGCGCGGAGGCCATGGCCCAGGCG ACCGATATCCTCGCTGACGGCGGCCTCCTGCGCGACCAGCGGTTCACACATCCAGCGGCCCGCTC GGTGGTGCTCGACGACATGTCCGCCGAGGAACGACGCAGCGTGCACAGCCTCGCCCTGGAACTGC TGGACGAGGCACCGGCCGAGATGCTCGCGCACCACCGGGTCGGCGCCGGTCTCGTGCACGGGCC GAAGGCCGCGGAGACATTCACCGGGGCCGGCCGGGCACTGGCCGTTCGCGGCATGCTGGGCGAG GCAGCCGACTACCTGCAACTGGCGTACCGGGCCTCCGGCGACGCCGCTACCAAGGCCGCGATACG CGTCGAGTCCGTGGCGGTCGAGCGCCGACGCAATCCGCTGGTCGTCAGTCGCCATTGGGACGAGC TGAGCGTCGCGGCCCGCGCCGGTCTGCTCTCCTGCGAGCACGTGTCCAGGACGGCCCGCTGGCTG ACCGTCGGTGGGCGGCCCGGCGAGGCGGCCAGGGTGCTGGCGTCGCAACACCGACGGGTCGTCA CCGATCAGGACCGGGCCCACCTGCGGGTCGCCGAGTTCTCGCTCGCGCTGCTGTACCCCGGTACG TCCGGCTCGGACCGGCGCCCGCACCCGCTCACGTCGGACGAACTCGCGGCCCTACCGACTGCGAC CAGACACTGCGCGATCGCCGATAACGCTGTCATGGCTGCCTTGCGTGGTCATCCGGAGCTTGCCAC CGCCGAGGCAGAAGCCGTTCTGCAGCAAGCCGACGCGGCGGACGGCGCTGCTCTCACCGCGCTG ATGGCCCTGCTGTACGCGGAGAGCATCGAGGTCGCTGAAGTCTGGGCGGACAAGCTGGCGGCAGA GGCCGGAGCATCGAACGGGCAGGACGCGGAGTACGCCGGTATACGCGCCGAAATCGCCCTGCGG CGCGGCGATCTGACCGCGGCCGTCGAGACCGCCGGCATGGTCCTGGACGGCCGGCCGCTGCCGT CGCTCGACATCACCGCCACGTTGCTGTTGGCCGGCAGGGCGTCCGTCGCCGTCCGGCTGGGCGAA CTCGACCACGCGGAGGAGCTGTTCGCCGCGCCGCCGGAGGACGCCTTCCAGGACAGCCTCTTCGG TCTGCATCTGCTCTCGGCGCACGGCCAGTACAGCCTCGCGACAGGCCGGCCCGAGTCGGCATACC GGGCCTTTCGTGCCTGCGGCGAACGTATGCGCGATTGGGGCTTCGACGCGCCCGGTGTGGCCCTG TGGCGCGTCGGCGCCGCCGAGGCGCTGCTCGGCCTCGACCGGAACGAGGGCCGACGGCTCATCG ACGAACAGCTGAGCCGGACGATGGCCCCCCGGTCCCACGCGTTGACGCTGCGGATAAAAGCGGCG TACATGCCGGAGCCGAAGCGGGTCGACCTGCTCTACGAAGCGGCTGAGCTGCTGCTCTCCTGCCG GGACCAGTATGAGCGAGCGCGGGTGCTCGCCGATCTGGGCGAGGCGCTCAGCGCGCTCGGGAAC TACCGGCAGGCGCGAGGTGTGCTCCGGCAGGCTCGGCATCTGGCCATGCGAACCGGCGCGGACC CGCTGCTGCGCCGGCTCGGAATCAGGCCCGGCCGGCAGGACGACCCCGACCCGCAGCCGCGGAG CAGATCGCTGACCAACGCTGAGCGGCGTGCGGCGTCGCTGGCCGCGACCGGACTGACCAACCGG GAGATCGCCGACCGGCTCTTCGTCACCGCCAGCACCGTGGAGCAGCACCTCACCAACGTCTTCCGC AAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGGCCGAGTTGGACGACATGGAATAG
SEQ ID NO: 62
ATGCGAGCTATTAATGCGTCCGACACCGGTCCTGAACTGGTCGCCCGCGAAGACGAACTGGGACGT GTACGAAGTGCCCTGAACCGAGCGAACGGCGGCCAAGGTGTCCTGATCTCCATTACCGGTCCGATC GCCTGCGGCAAGACCGAACTGCTTGAGGCTGCCGCCTCGGAAGTTGACGCCATCACTCTGCGCGC GGTCTGTGCCGCCGAGGAACGGGCGATACCTTATGCCCTGATCGGGCAGCTTATCGACAACCCCGC GCTCGGCATTCCGGTTCCGGATCCGGCCGGCCTGACCGCCCAGGGCGGACGACTGTCATCGAGCG CCGAGAACCGACTGCGTCGCGACCTCACCCGTGCCCTGCTGACGCTCGCCACCGACCGGCTGGTG CTGATCTGTGTCGATGACGTGCAGCACGCCGACAACGCCTCGTTGAGCTGCCTTCTGTATCTGGCC CGACGGCTTGTCCCGGCTCGAATCGCTCTGGTATTCACCGAGTTGCGAGTCCTCACCTCGTCTCAG CTGCGGTTCAACGCGGAGCTGCTCAGCTTGCGGAACCACTGCGAGATCGCGCTGCGCCCACTCGG CCCGGGGCATGCGGCCGAGCTGGCCCGCGCCACCCTCGGCCCCGGCCTCTCCGACGAAACACTC ACGGAGCTGTACCGGGTGACCGGAGGCAACCTGAGTCTCAGCCGCGGGCTGATCGACGATGTGCG GGACGCCTGGGCACGAGGGGAAACGGGCGTCCAGGTGGGCCGGGCGTTCCGGCTGGCCTACCTC GGTTCCCTCCACCGCTGTGGTCCGCTGGCGTTGCGGGTCGCCCGCGTAGCCGCCGTACTGGGCCC GAGCGCCACCAGCGTCCTGGTGCGCCGGATCAGTGGGCTCAGCGCGGAGGCCATGGCCCAGGCG ACCGATATCCTCGCTGACGGCGGCCTCCTGCGCGACCAGCGGTTCACACATCCAGCGGCCCGCTC GGTGGTGCTCGACGACATGTCCGCCGAGGAACGACGCAGCGTGCACAGCCTCGCCCTGGAACTGC TGGACGAGGCACCGGCCGAGATGCTCGCGCACCACCGGGTCGGCGCCGGTCTCGTGCACGGGCC GAAGGCCGCGGAGACATTCACCGGGGCCGGCCGGGCACTGGCCGTTCGCGGCATGCTGGGCGAG GCAGCCGACTACCTGCAACTGGCGTACCGGGCCTCCGGCGACGCCGCTACCAAGGCCGCGATACG CGTCGAGTCCGTGGCGGTCGAGCGCCGACGCAATCCGCTGGTCGTCAGTCGCCATTGGGACGAGC TGAGCGTCGCGGCCCGCGCCGGTCTGCTCTCCTGCGAGCACGTGTCCAGGACGGCCCGCTGGCTG ACCGTCGGTGGGCGGCCCGGCGAGGCGGCCAGGGTGCTGGCGTCGCAACACCGACGGGTCGTCA CCGATCAGGACCGGGCCCACCTGCGGGTCGCCGAGTTCTCGCTCGCGCTGCTGTACCCCGGTACG TCCGGCTCGGACCGGCGCCCGCACCCGCTCACGTCGGACGAACTCGCGGCCCTACCGACTGCGAC CAGACACTGCGCGATCGCCGATAACGCTGTCATGGCTGCCTTGCGTGGTCATCCGGAGCTTGCCAC CGCCGAGGCAGAAGCCGTTCTGCAGCAAGCCGACGCGGCGGACGGCGCTGCTCTCACCGCGCTG ATGGCCCTGCTGTACGCGGAGAGCATCGAGGTCGCTGAAGTCTGGGCGGACAAGCTGGCGGCAGA GGCCGGAGCATCGAACGGGCAGGACGCGGAGTACGCCGGTATACGCGCCGAAATCGCCCTGCGG CGCGGCGATCTGACCGCGGCCGTCGAGACCGCCGGCATGGTCCTGGACGGCCGGCCGCTGCCGT CGCTCGACATCACCGCCACGTTGCTGTTGGCCGGCAGGGCGTCCGTCGCCGTCCGGCTGGGCGAA CTCGACCACGCGGAGGAGCTGTTCGCCGCGCCGCCGGAGGACGCCTTCCAGGACAGCCTCTTCGG TCTGCATCTGCTCTCGGCGCACGGCCAGTACAGCCTCGCGACAGGCCGGCCCGAGTCGGCATACC GGGCCTTTCGTGCCTGCGGCGAACGTATGCGCGATTGGGGCTTCGACGCGCCCGGTGTGGCCCTG TGGCGCGTCGGCGCCGCCGAGGCGCTGCTCGGCCTCGACCGGAACGAGGGCCGACGGCTCATCG ACGAACAGCTGAGCCGGACGATGGCCCCCCGGTCCCACGCGTTGACGCTGCGGATAAAAGCGGCG TACATGCCGGAGCCGAAGCGGGTCGACCTGCTCTACGAAGCGGCTGAGCTGCTGCTCTCCTGCCG GGACCAGTATGAGCGAGCGCGGGTGCTCGCCGATCTGGGCGAGGCGCTCAGCGCGCTCGGGAAC TACCGGCAGGCGCGAGGTGTGCTCCGGCAGGCTCGGCATCTGGCCATGCGAACCGGCGCGGACC CGCTGCTGCGCCGGCTCGGAATCAGGCCCGGCCGGCAGGACGACCCCGACCCGCAGCCGCGGAG CAGATCGCTGACCAACGCTGAGCGGCGTGCGGCGTCGCTGGCCGCGACCGGACTGACCAACCGG GAGATCGCCGACCGGCTCTTCGTCACCGCCAGCACCGTGGAGCAGCACCTCACCAACGTCTTCCGC AAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGGCCGAGTTGGACGACATGGAATAG SEQ ID NO: 63
MPAVECYELDARDDELRKLEEVVTGRANGRGVVVTITGPIACGKTELLDAAAAKADAITLRAVCSAEEQAL PYALIGQLIDNPALASHALEPACPTLPGEHLSPEAENRLRSDLTRTLLALAAERPVLIGIDESHANALCLLHL ARRVGSARIAMVLTELRRLTPAHSQFQAELLSLGHHREIALRPLSPKHTAELVRAGLGPDVDEDVLTGLYR ATGGNLNLTRGLINDVREAWETGGTGISAGRAYRLAYLGSLYRCGPVPLRVARVAAVLGQSANTTLVRWI SGLNADAVGEATEILTEGGLLHDLRFPHPAARSVVLNDMSAQERRRLHRSALEVLDDVPVEVVAHHQVG AGLLHGPKAAEIFAKAGQELHVRGELDTASDYLQLAHQASDDAVTGMRAEAVAIERRRNPLASSRHLDEL TVVARAGLLFPEHTALMIRWLGVGGRSGEAAGLLASQRPRAVTDQDRAHMRAAEVSLALVSPGTSGPD RRPRPLTPDELANLPKAARLCAIADNAVMSALRGRPELAAAEAENVLQHADSAAAGTTALAALTALLYAE NTDTAQLWADKLVSETGASNEEEAGYAGPRAEAALRRGDLAAAVEAGSTVLDHRRLSTLGITAALPLSSA VAAAIRLGETERAEKWLAQPLPQAIQDGLFGLHLLSARGQYSLATGQHESAYTAFRTCGERMRNWGVDV PGLSLWRVDAAEALLHGRDRDEGRRLVDEQLTRAMGPRSRALTLRVQAAYSPPAKRVDLLDEAADLLLS CNDQYERARVLADLSETFSALRHHSRARGLLRQARHLAAQRGAIPLLRRLGAKPGGPGWLEESGLPQRI KSLTDAERRVASLAAGGQTNRVIADQLFVTASTVEQHLTDVSTGSRPPAPAAELV SEQ ID NO: 64
MVPEVRAAPDELIARDDELSRLQRALTRAGSGRGGVVAITGPIASGKTALLDAGAAKSGFVALRAVCSWE ERTLPYGMLGQLFDHPELAAQAPDLAHFTASCESPQAGTDNRLRAEFTRTLLALAADWPVLIGIDDVHHA DAESLRCLLHLARRIGPARIAVVLTELRRPTPADSRFQAELLSLRSYQEIALRPLTEAQTGELVRRHLGAET HEDVSADTFRATGGNLLLGHGLINDIREARTAGRPGVVAGRAYRLAYLSSLYRCGPSALRVARASAVLGA SAEAVLVQRMTGLNKDAVEQVYEQLNEGRLLQGERFPHPAARSIVLDDLSALERRNLHESALELLRDHG VAGNVLARHQIGAGRVHGEEAVELFTGAAREHHLRGELDDAAGYLELAHRASDDPVTRAALRVGAAAIE RLCNPVRAGRHLPELLTASRAGLLSSEHAVSLADWLAMGGRPGEAAEVLATQRPAADSEQHRALLRSG ELSLALVHPGAWDPLRRTDRFAAGGLGSLPGPARHRAVADQAVIAALRGRLDRADANAESVLQHTDATA DRTTAIMALLALLYAENTDAVQFWVDKLAGDEGTRTPADEAVHAGFNAEIALRRGDLMRAVEYGEAALG HRHLPTWGMAAALPLSSTVVAAIRLGDLDRAERWLAEPLPQQTPESLFGLHLLWARGQHHLATGRHGAA YTAFRECGERMRRWAVDVPGLALWRVDAAESLLLLGRDRAEGLRLVSEQLSRPMRPRARVQTLRVQAA YSPPPQRIDLLEEAADLLVTCNDQYELANVLSDLAEASSMVRQHSRARGLLRRARHLATQCGAVPLLRRL GAEPSDIGGAWDATLGQRIASLTESERRVAALAAVGRTNREIAEQLFVTASTVEQHLTNVFRKLAVKGRQ QLPKELADVGEPADRDRRCG SEQ ID NO: 65
MIARLSPPDLIARDDEFGSLHRALTRAGGGRGVVAAVTGPIACGKTELLDAAAAKAG FVTLRAVCSMEER ALPYGMLGQLLDQPELAARTPELVRLTASCENLPADVDNRLGTELTRTVLTLAAERPVLIGIDDVHHADAP SLRCLLHLARRISRARVAIVLTELLRPTPAHSQFRAALLSLRHYQEIALRPLTEAQTTELVRRHLGQDAHDD VVAQAFRATGGNLLLGHGLIDDIREARTRTSGCLEVVAGRAYRLAYLGSLYRCGPAALSVARASAVLGES VELTLVQRMTGLDTEAVEQAHEQLVEGRLLREGRFPHPAARSVVLDDLSAAERRGLHELALELLRDRGV ASKVLARHQMGTGRVHGAEVAGLFTDAAREHHLRGELDEAVTYLEFAYRASDDPAVHAALRVDTAAIER LCDPARSGRHVPELLTASRERLLSSEHAVSLACWLAMDGRPGEAAEVLAAQRSAAPSEQGRAHLRVAD LSLALIYPGAADPPRPADPPAEDEVASFSGAVRHRAVADKALSNALRGWSEQAEAKAEYVLQHSRVTTD RTTTMMALLALLYAEDTDAVQSWVDKLAGDDNMRTPADEAVHAGFRAEAALRRGDLTAAVECGEAALA PRVVPSWGMAAALPLSSTVAAAIRLGDLDRAERWLAEPLPEETSDSLFGLHMVWARGQHHLAAGRYRA AYNAFRDCGERMRRWSVDVPGLALWRVDAAEALLLLGRGRDEGLRLISEQLSRPMGSRARVMTLRVQA AYSPPAKRIELLDEAADLLIMCRDQYELARVLADMGEACGMLRRHSRARGLFRRARHLATQCGAVPLLR RLGGESSDADGTQDVTPAQRITSLTEAERRVASHAAVGRTNKEIASQLFVTSSTVEQHLTNVFRKLGVKG RQQLPKELSDAG
SEQ ID NO: 66
MEFYDLVARDDELRRLDQALGRAAGGRGVVVTVTGPVGCGKTELLDAAAAEEEFITLRAVCSAEERALP YAVIGQLLDHPVLSARAPDLACVTAPGRTLPADTENRLRRDLTRALLALASERPVLICIDDVHQADTASLN CLLHLARRVASARIAMILTELRRLTPAHSRFEAELLSLRHRHEIALRPLGPADTAELARARLGAGVTADELA QVHEATSGNPNLVGGLVNDVREAWAAGGTGIAAGRAYRLAYLSSVYRCGPVPLRIAQAAAVLGPSATVT LVRRISGLDAETVDEATAILTEGGLLRDHRFPHPAARSVVLDDMSAQERRRLHRSTLDVLDGVPVDVLAH HQAGAGLLHGPQAAEMFARASQELRVRGELDAATEYLQLAYRASDDAGARAALQVETVAGERRRNPLA ASRHLDELAAAARAGLLSAEHAALVVHWLADAGRPGEAAEVLALQRALAVTDHDRARLRAAEVSLALFH PGVPGSDPRPLAPEELASLSLSARHGVTADNAVLAALRGRPESAAAEAENVLRNADAAASGPTALAALTA LLYAENTDAAQLWADKLAAGIGAGEGEAGYAGPRTVAALRRGDLTTAVQAAGAVLDRGRPSSLGITAVLP LSGAVAAAIRLGELERAEKWLAEPLPEAVHDSLFGLHLLMARGRYSLAVGRHEAAYAAFRDCGERMRRW DVDVPGLALWRVDAAEALLPGDDRAEGRRLIDEQLTRPMGPRSRALTLRVRAAYAPPAKRIDLLDEAADL LLSSNDQYERARVLADLSEAFSALRQNGRARGILRQARHLAAQCGAVPLLRRLGVKAGRSGRLGRPPQG IRSLTEAERRVATLAAAGQTNREIADQLFVTASTVEQHLTNVFRKLGVKGRQQLPAELADLRPPG SEQ ID NO: 67
MYSGTCREGYELVAREDELGILQRSLEQASSGQGVVVTVTGPIACGKTELLDAAAAKAEAIILRAVCAPEE RAMPYAMIGQLIDDPALAHRAPGLADRIAQGGQLSLRAENRLRRDLTRALLALAVDRPVLIGVDDVHHAD TASLNCLLHLARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPFGPEQSAELARAAFGPGLA EDVLVGLYKTTRGNLSLSRGLISDVREALANGESAFEAGRAFRLAYLGSLYRCGPVALRVARVAAVLGPS ATTTLVRRLSGLSAETIDRATKILTEGGLLLDQQFPHPAARSVVLDDMSAQERRGLHTLALELLDEAPVEV LAHHQVGAGLIHGPKAAEMFAKAGKALVVRNELGDAAEYLQLAHRASDDVSTRAALRVEAVAIERRRNPL ASSRHMDELSAAGRAGLLSPKHAALAVFWLADGGRSGEAAEVLASERPLATTDQNRAHLRFVEVTLALF SPGAFGSDRRPPPLTPDELASLPKAAWQCAVADNAAMTALHGHPELATAQAETVLRQADSAADAIPAALI ALLYAENTESAHIWADKLGSTNGGVSNEAEAGYAGPCAEIALRRGDLATAFEAGSTVLDDRSLPSLGITAA LLLSSKTAAAVRLGELERAEKLLAEPLPNGVQDSLFGLHLLSAYGQYSLAMGRYESALRAFHTCGERMRS WDVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLTRPMGPRSRALTLRIKAAYLPRTKRIPLLHEAAELL LPCPDPYEQARVLADLGDTLSALRRYSRARGVLRQARHLAAQCGAVPLLRRLGGEPGRIDDAGLPQRST SLTDAERRVAALAAAGQTNREIAKQLFVTASTVEQHLTSVFRKLGVKGRKQLPTALADVEQT
SEQ ID NO: 68
MPAVESYELDARDDELRRLEEAVGQAGNGRGVVVTITGPIACGKTELLDAAAAKSDAITLRAVCSEEERA
LPYALIGQLIDNPAVASQLPDPVSMALPGEHLSPEAENRLRGDLTRTLLALAAERPVLIGIDDMHHADTASL
NCLLHLARRVGPARIAMVLTELRRLTPAHSQFHAELLSLGHHREIALRPLGPKHIAELARAGLGPDVDEDV LTGLYRATGGNLNLGHGLIKDVREAWATGGTGINAGRAYRLAYLGSLYRCGPVPLRVARVAAVLGQSAN TTLVRWISGLNADAVGEATEILTEGGLLHDLRFPHPAARSVVLNDLSARERRRLHRSALEVLDDVPVEVVA HHQAGAGFIHGPKAAEIFAKAGQELHVRGELDAASDYLQLAHHASDDAVTRAALRVEAVAIERRRNPLAS SRHLDELTVAARAGLLSLEHAALMIRWLALGGRSGEAAEVLAAQRPRAVTDQDRAHLRAAEVSLALVSP GASGVSPGASGPDRRPRPLPPDELANLPKAARLCAIADNAVISALHGRPELASAEAENVLKQADSAADGA TALSALTALLYAENTDTAQLWADKLVSETGASNEEEGAGYAGPRAETALRRGDLAAAVEAGSAILDHRRG SLLGITAALPLSSAVAAAIRLGETERAEKWLAEPLPEAIRDSLFGLHLLSARGQYCLATGRHESAYTAFRTC GERMRNWGVDVPGLSLWRVDAAEALLHGRDRDEGRRLIDEQLTHAMGPRSRALTLRVQAAYSPQAQR VDLLEEAADLLLSCNDQYERARVLADLSEAFSALRHHSRARGLLRQARHLAAQCGATPLLRRLGAKPGG PGWLEESGLPQRIKSLTDAERRVASLAAGGQTNRVIADQLFVTASTVEQHLTNVFRKLGVKGRQHLPAEL ANAE
SEQ ID NO: 69
MPAVKRNDLVARDGELRWMQEILSQASEGRGAVVTITGAIACGKTVLLDAAAASQDVIQLRAVCSAEEQE LPYAMVGQLLDNPVLAARVPALGNLAAAGERLLPGTENRIRRELTRTLLALADERPVLIGVDDMHHADPA SLDCLLHLARRVGPARIAIVLTELRRLTPAHSRFQSELLSLRYHHEIGLQPLTAEHTADLARVGLGAEVDDD VLTELYEATGGNPSLCCGLIRDVRQDWEAGVTGIHVGRAYRLAYLSSLYRCGPAALRTARAAAVLGDSA DACLIRRVSGLGTEAVGQAIQQLTEGGLLRDQQFPHPAARSVVLDDMSAQERHAMYRSAREAAAEGQA DPGTPGEPRAATAYAGCGEQAGDYPEPAGRACVDGAG PAEYCGDPHGADDDPDELVAALGGLLPSRL VAMKIRRLAVAGRPGAAAELLTSQRLHAVTSEDRASLRAAEVALATLWPGATGPDRHPLTEQEAASLPE GPRLLAAADDAVGAALRGRAEYAAAEAENVLRHADPAAGGDAYAAMIALLYTEHPENVLFWADKLDAGR PDEETSYPGLRAETAVRLGDLETAMELGRTVLDQRRLPSLGVAAGLLLGGAVTAAIRLGDLDRAEKWLAE PIPDAIRTSLYGLHVLAARGRLDLAAGRYEAAYTAFRLCGERMAGWDADVSGLALWRVDAAEALLSAGIR PDEGRKLIDDQLTREMGARSRALTLRAQAAYSLPVHRVGLLDEAAGLLLACHDGYERARVLADLGETLRT LRHTDAAQRVLRQAEQAAARCGSVPLLRRLGAEPVRIGTRRGEPGLPQRIRLLTDAERRVAAMAAAGQT NREIAGRLFVTASTVEQHLTSVFRKLGVKGRRFLPTELAQAV
SEQ ID NO: 70
MYSGTCREGYELVAREDELGILQRSLEQASSGQGVVVTVTGPIACGKTELLDAAAAKAEAIILRAVCAPEE
RAMPYAMIGQLIDDPALAHRAPGLADRIAQGGQLSLRAENRLRRDLTRALLALAVDRPVLIGVDDVHHAD
TASLNCLLHLARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPFGPEQSAELARAAFGPGLA EDVLAGLYKTTRGNLSLSRGLISDVREALANGESAFEAGRAFRLAYLSSLYRCGPVALRVARVAAVLGPS ATTTLVRRLSGLSAETIDRATKILTEGGLLLDQQFPHPAARSVVLDDMSAQERRSLHTLALELLDEAPVEVL AHHQVGAGLIHGPKAAEMFAKAGKALVVRNELGDAAEYLQLAHRASDDVSTRAALRVEAVAIERRRNPL ASSRHMDELSAAGRAGLLSPKHAALAVFWLADGGRSGEAAEVLASERPLATTDQNRAHLRFVEVTLALF SPGAFGSDRRPPPLTPDELASLPKAAWQCAVADNAAMTALHGHPELATAQAETVLRQADSAADAIPAALI ALLYAENTESAHIWADKLGSTNAGVSNEAEAGYAGPCAEIALRRGDLATAFEAGSAVLDDRSLPSLGITAA LLLSSKTAAAVRLGELERAEKLLAEPLPNGVQDSLFGLHLLSAYGQYSLAMGRYESAHRAFRTCGERMR SWDVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLTRPMGPRSHALTLRIKAAYLPRTKRIPLLHEAAEL LLPCPDPYEQARVLADLGDTLSALRRYSRARGVLRQARHLATQCGAVPLLRRLGGEPGRIDDAGLPQRS TSLTDAERRVAALAAAGQTNREIAEQLFVTASTVEQHLTSVFRKLGVKGRKQLPTALADVEQT
SEQ ID NO: 71
MYSGTCREGYELVAREDELGILQRSLEEAGSGQGAVVTVTGPIACGKTELLDAAAAKADAIILRAVCAPEE RAMPYAMIGQLIDDPALAHRAPELADRIAQGGHLSLRAENRLRRDLTRALLALAVDRPVLIGVDDVHHADT ASLNCLLHLARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPLGPEQSAELAHAAFGPGLAE DVLAGLYGMTRGNLSLSRGLISDVREAQANGESAFEVGRAFRLAYLSSLYRCGPIALRVARVAAVLGPSA TTTLVRRLSGLSAETIDRATKILTEGGLLLDHQFPHPAARSVVLDDMSAQERRSLHTLALELLDEAPVEVLA HHQVGAGLIHGPKAAEIFARAGQALVVRNELGDAAEYLQLAHRASDDVSTRAALRVEAVAIERRRNPLAS SRHMDELSAAGRAGLLSPKHAALAVFWLADGGRSGEAAEVLASEHPLATTDQNRAHLRFAEVTLALFCP GAFGSDRRPPPLAPDELASLPKAAWQCAVADNAVMTALHAHPELATAQAETVLRQADSAADAIPAALIAL LYAENTESAQIWADKLGSTNAGVSNEAEAGYAGPCAEIALRRGDLATAFEAGGTVLDDRPLPSLGITAALL LSSKTAAAVRLGELERAEKLLAEPLPNGVQDSLFGLHLLSAHGQYSLAMGRYESAHRAFHTCGERMRSW GVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLARPMGPRSRALTLRIKAAYLPRTKRIPLLHEAAELLL SCPDPYEQARVLADLGDTLSALRRYSRARGVLRQARHLATQCGAVPLLRRLGGEPGRIDDAGLPQRSTS LTDAERRVSALAAAGQTNREIAKQLFVTASTVEQHLTSVFRKLGVKGRRQLPTALADVE
SEQ ID NO: 72
MYSGTCREGYELVAREDELGILQRSLEQASSGQGVVVTVTGPIACGKTELLDAAAAKAEAIILRAVCAPEE RAMPYAMIGQLIDDPALAHRAPGLADRIAQGGQLSLRAENRLRRDLTRALLALAVHRPVLIGVDDVHHAD TASLNCLLHLARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPFGPEQSAELARAAFGPGLA EDVLAGLYKTTRGNLSLSRGLISDVREALANGESAFEAGRAFRLAYLSSLYRCGPVALRVARVAAVLGPS ATTTLVRRLSGLSAETIDRATKILTEGGLLLDQQFPHPAARSVVLDDMSAQERRGLHTLALELLDEAPVEV LAHHQVGAGLIHGPKAAEMFAKAGKALVVRNELGDAAEYLQLAHRASDDVSTRAALRVEAVAIERRRNPL ASSRHMDELSAAGRAGLLSPKHAALAVFWLADGGRSGEAAQVLASERPLATTDQNRAHLRFVEVTLALF SPGAFGSDRRPPPLTPDELASLPKAAWQCAVADNAAMTALHGHPELATAQAETVLRQADSAADAIPAALI ALLYAENTESAHIWADKLGSMNAGVSNEAEAGYAGPCAEIALRRGDLATAFEAGSTVLDDRSLPSLGITA ALLLSSKTAAAVRLGELERAEKLLAEPLPNGVQDSLFGLHLLSAYGQYSLAMGRYESAHRAFRTCGERM RSWDVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLTRPMGPRSRALTLRIKAAYLPRTKRIPLLHEAA ELLLPCPDPYEQARVLADLGDTLSALRRYSRARGVLRQARHLATQCGAVPLLRRLGGEPGRIDDAGLPQ RSTSLTDAERRVAALAAAGQTNREIAEQLFVTASTVEQHLTSVFRKLGVKGRKQLPTALADVEQT SEQ ID NO: 73
MRAINASDTGPELVAREDELGRVRSALNRANGGQGVLISITGPIACGKTELLEAAASEVDAITLRAVCAAE ERAIPYALIGQLIDNPALGIPVPDPAGLTAQGGRLSSSAENRLRRDLTRALLTLATDRLVLICVDDVQHADN ASLSCLLYLARRLVPARIALVFTELRVLTSSQLRFNAELLSLRNHCEIALRPLGPGHAAELARATLGPGLSD ETLTELYRVTGGNLSLSRGLIDDVRDAWARGETGVQVGRAFRLAYLGSLHRCGPLALRVARVAAVLGPS ATSVLVRRISGLSAEAMAQATDILADGGLLRDQRFTHPAARSVVLDDMSAEERRSVHSLALELLDEAPAE MLAHHRVGAGLVHGPKAAETFTGAGRALAVRGMLGEAADYLQLAYRASGDAATKAAIRVESVAVERRR NPLVVSRHWDELSVAARAGLLSCEHVSRTARWLTVGGRPGEAARVLASQHRRVVTDQDRAHLRVAEFS LALLYPGTSGSDRRPHPLTSDELAALPTATRHCAIADNAVMAALRGHPELATAEAEAVLQQADAADGAAL TALMALLYAESIEVAEVWADKLAAEAGASNGQDAEYAGIRAEIALRRGDLTAAVETAGMVLDGRPLPSLDI TATLLLAGRASVAVRLGELDHAEELFAAPPEDAFQDSLFGLHLLSAHGQYSLATGRPESAYRAFRACGER MRDWGFDAPGVALWRVGAAEALLGLDRNEGRRLIDEQLSRTMAPRSHALTLRIKAAYMPEPKRVDLLYE AAELLLSCRDQYERARVLADLGEALSALGNYRQARGVLRQARHLAMRTGADPLLRRLGIRPGRQDDPDP QPRSRSLTNAERRAASLAATGLTNREIADRLFVTASTVEQHLTNVFRKLGVKGRKQLPAELDDME LAL Binding Sites
In some embodiments, a gene cluster (e.g., a PKS gene cluster) includes one or more promoters that include one or more LAL binding sites. The LAL binding sites may include a polynucleotide consensus LAL binding site sequence (e.g., as described herein). In some instances, the LAL binding site includes a core AGGGGG (SEQ ID NO: 74) motif. In certain instances, the LAL binding site includes a sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) homology to SEQ ID NO: 39. The LAL binding site may include mutation sites that have been restored to match the sequence of a consensus or optimized LAL binding site. In some embodiments, the LAL binding site is a synthetic LAL binding site. In some embodiments, synthetic LAL binding sites may be identified by (a) providing a plurality of synthetic nucleic acids including at least eight nucleotides; (b) contacting one or more of the plurality of nucleotides including at least eight nucleotides with one or more LALs; (c) determining the binding affinity between a nucleic acid of step (a) and an LAL of step (b), wherein a synthetic nucleic acid is identified as a synthetic LAL binding site if the affinity between the synthetic nucleic acid and an LAL is greater than X. The identified synthetic LAL binding sites may then be introduced into a host cell in a compound-producing cluster (e.g., a PKS cluster).
In some embodiments, a pair of LAL binding site and a heterologous LAL or a heterologous LAL binding site and an LAL that have increased expression compared to a natural pair may be identified by (a) providing one or more LAL binding sites; (b) contacting one or more of the LAL binding sites with one or more LALs; (c) determining the binding affinity between a LAL binding site and an LAL, wherein a pair having increased expression is identified if the affinity between the LAL binding site and the LAL is greater than the affinity between the LAL binding site and its homologous LAL and/or the LAL at its homologous LAL binding site. In some embodiments, the binding affinity between the LAL binding site and the LAL is determined by determining the expression of a protein or compound by a cell which includes both the LAL and the LAL binding site.
Constitutively active LALs
In some embodiments, the recombinant LAL is a constitutively active LAL. For example, the amino acid sequence of the LAL has been modified in such a way that it does not require the presence of an inducer compound for the altered LAL to engage its cognate binding site and activate transcription of a compound producing protein (e.g., polyketide synthase). Introduction of a constitutively active LAL to a host cell would likely result in increased expression of the compound-producing protein (e.g., polyketide synthase) and, in turn, increased production of the corresponding compound (e.g., polyketide).
Engineering Unidirectional LALs
FkPhD gene clusters are arranged with a multicistronic architecture driven by multiple bidirectional promoter-operators that harbor conserved (in single or multiple, and inverted to each other and/or directly repeating) GGGGGT (SEQ ID NO: 40) motifs presumed to be LAL binding sites.
Bidirectional LAL promoters may be converted to unidirectional ones (UniLALs) by strategically deleting one of the opposing promoters, but maintaining the tandem LAL binding sites (in case binding of LALs in the native promoter is cooperative, as was demonstrated for MalT). Functionally this is achieved by removal of all sequences 3' of the conserved GGGGGT (SEQ ID NO: 40) motif present on the antisense strand (likely containing the -35 and -10 promoter sequences), but leaving intact the entire sequence on the sense strand. As a consequence of this deletion, transcription would be activated in one direction only. The advantages of this feed-forward circuit architecture would be to tune and/or maximize LAL expression during the complex life cycle of Streptomyces vegetative and fermentation growth conditions. Host cells
In some embodiments, the host cell is a bacteria such as an Actiobacterium. For example, in some embodiments, the host cell is a Streptomyces strain. In some embodiments, the host cell is Streptomyces anulatus, Streptomyces antibioticus, Streptomyces coelicolor, Streptomyces peucetius, Streptomyces sp. ATCC 700974, Streptomyces canus, Streptomyces nodosus, Streptomyces (multiple sp.), Streptoalloteicus hindustanus, Streptomyces hygroscopicus, Streptomyces avermitilis, Streptomyces viridochromogenes, Streptomyces verticillus, Streptomyces chartruensis, Streptomyces (multiple sp.), Saccharothrix mutabilis, Streptomyces halstedii, Streptomyces clavuligerus, Streptomyces venezuelae, Strteptomyces roseochromogenes, Amycolatopsis orientalis, Streptomyces clavuligerus, Streptomyces rishiriensis, Streptomyces lavendulae, Streptomyces roseosporus, Nonomuraea sp., Streptomyces peucetius, Saccharopolyspora erythraea, Streptomyces filipinensis, Streptomyces hygroscopicus, Micromonospora purpurea, Streptomyces hygroscopicus, Streptomyces narbonensis, Streptomyces kanamyceticus, Streptomyces collinus, Streptomyces lasaliensis, Streptomyces lincolnensis, Dactosporangium aurantiacum, Streptomyces toxitricini, Streptomyces hygroscopicus, Streptomyces plicatus, Streptomyces lavendulae, Streptomyces ghanaensis, Streptomyces cinnamonensis,
Streptomyces aureofaciens, Streptomyces natalensis, Streptomyces chattanoogensis L 10, Streptomyces lydicus A02, Streptomyces fradiae, Streptomyces ambofaciens, Streptomyces tendae, Streptomyces noursei, Streptomyces avermitilis, Streptomyces rimosus, Streptomyces wedmorensis, Streptomyces cacaoi, Streptomyces pristinaespiralis, Streptomyces pristinaespiralis, Actinoplanes sp. ATCC 33076, Streptomyces hygroscopicus, Lechevalieria aerocolonegenes, Amycolatopsis mediterranei,
Amycolatopsis lurida, Streptomyces albus, Streptomyces griseolus, Streptomyces spectabilis,
Saccharopolyspora spinosa, Streptomyces ambofaciens, Streptomyces staurosporeus, Streptomyces griseus, Streptomyces (multiple species), Streptomyces acromogenes, Streptomyces tsukubaensis, Actinoplanes teichomyceticus, Streptomyces glaucescens, Streptomyces rimosus, Streptomyces cattleya, Streptomyces azureus, Streptoalloteicus hindustanus, Streptomyces chartreusis, Streptomyces fradiae, Streptomyces coelicolor, Streptomyces hygroscopicus, Streptomyces sp. 1 1861, Streptomyces virginiae, Amycolatopsis japonicum, Amycolatopsis balhimycini, Streptomyces albus J 1074, Streptomyces coelicolor M1 146, Streptomyces lividans, Streptomyces incarnates, Streptomyces violaceoruber, or Streptomyces griseofuscus. In some embodiments, the host cell is an Escherichia strain such as Escherichia coli. In some embodiments, the host cell is a Bacillus strain such as Bacillus subtilis. In some embodiments, the host cell is a Pseudomonas strain such as Pseudomonas putitda. In some embodiments, the host cell is a Myxococcus strain such as Myxococcus xanthus.
Methods
The proteins, nucleic acids, vectors, and host cells of the invention may be used for production of compounds (e.g., polyketides). Introduction of heterologous domains to proteins allow alteration of the chemical structure of polyketides produced by the proteins.
Introduction of heterologous domains
The activity of β-ketone processing domains can be altered by introducing the sequences of domains from other polyketide synthases. Multiple heterologous sequences can be tested for their ability to alter the activity of a specific domain without drastically reducing the amount of polyketide expressed. New variants of the polyketide synthase can be subjected to rigorous quality control (Sanger sequencing of region of interest, PCR-based "tiling" to confirm cluster integrity and lllumina sequencing to sequence the entire BAC). BACs may then be conjugated to two optimized Streptomyces producer strains, and solid-phase extracted (SPE) samples can be subjected to Top-Down mass spectrometry with purified FKBP12 protein to identify produced compounds.
A representative example of a workflow to generate a compound from a chimeric polyketide synthase includes grafting a short peptide sequence from the domain of one polyketide synthase, e.g., a ketoreductase domain, onto another polyketide synthase using homology-based cloning. For example, the catalytic Tyr of one ketoreductase may be replaced with Phe and the active site aFG loop may also deleted to inactive the domain. The resulting clone may then be conjugated into a Streptomyces expression host and fermented. Compounds may then be identified using comparative LC-TOF analysis of unfractionated SPE samples. Top Down mass spectrometry analysis may also be performed by co- injecting purified native FKBP12 and a compound from the modified polyketide synthase with a compound from the unmodified polyketide synthase. This analysis can show a mass difference between the two compounds consistent with the change in activity of the domain, e.g., a difference of 2 for an inactivated ketoreductase domain.
Compounds with multiple structural changes may be generated using combinations of KR, DH or ER single variants.
Production of libraries of engineered polyketide synthases
Combinatorial domain level engineering may be performed by combining multiple domain-level variants on a single protein backbone, thus enabling library-scale construction of diverse PKS/NRPS molecules for drug development.
Alternately, multiplex parallel engineering (e.g., by site-directed mutagenesis) may be used to produce libraries of engineered PKS/NRPS molecules for drug development. For example, site-directed mutagenesis of a polynucleotide encoding a parent polyketide synthase may be used to generate, in parallel, a plurality of polynucleotides encoding a plurality of engineered polyketide synthases. In some embodiments, each of the plurality of engineered polyketide synthases includes at least one codon modification relative to of the parent polyketide synthase (e.g., a codon that specifies a residue in a conserved motif of at least one domain of the parent polyketide synthase).
Characterization of engineered PKS libraries by single-molecule long-read sequencing
In some embodiments of the invention, single-molecule long-read sequencing technology (e.g., Nanopore sequencing or SMRT sequencing) may be used to characterize libraries of engineered polyketide synthases or non-ribosomal peptide synthases which are produced by any of the methods described herein. In particular, single-molecule long-read sequencing (e.g., Nanopore sequencing or
SMRT sequencing) may be used to characterize (e.g., deconvolute) combinatorial or multiplex libraries of engineered polyketide synthases or non-ribosomal peptide synthases (e.g., multiplex libraries generated by parallel engineering). Single-molecule long-read sequencing enables the identification of the module or modules which are incorporated into the combinatorial library. This further enables the prediction of the chemistry of the resulting plurality of engineered polyketide synthases or non-ribosomal peptide synthases. The predicted enzymatic chemistry can therefore be connected to the compounds produced by the engineered polyketide synthases or non-ribosomal peptide synthases. The resulting compounds may be identified by chemical methods of analysis known to one of skill in the art (e.g., mass
spectrometry or high performance liquid chromatography). Furthermore, the predicted enzymatic chemistry can be connected to the function of the resulting compounds (e.g., binding to a target protein or inducing a phenotype, such as a cell based phenotype). Accordingly, long-read sequencing of a genetically encoded molecule may allow for genotypic-phenotypic linkage.
Single-molecule long-read sequencing technologies may be considered to include any sequencing technology which enables the sequencing of a single molecule of a biopolymer (e.g., a polynucleotide such as DNA or RNA), and which enables read lengths of greater than 2 kilobases (e.g., greater than 5 kilobases, greater than 10 kilobases, greater than 20 kilobases, greater than 50 kilobases, or greater 100 kilobases). Single-molecule long-read sequencing technologies may enable the sequencing of multiple single molecules of DNA or RNA in parallel. Single-molecule long-read sequencing technologies may include sequencing technologies that rely on individual
compartmentalization of each molecule of DNA or RNA being sequenced.
Nanopore sequencing is an exemplary single-molecule long-read sequencing technology that may be used to characterize libraries of engineered polyketide synthases or non-ribosomal peptide synthases that are prepared by any of the methods described herein. Nanopore sequencing enables the long-read sequencing of single molecules of biopolymers (e.g., polynucleotides such as DNA or RNA). Nanopore sequencing relies on protein nanopores set in an electrically resistant polymer membrane. An ionic current is passed through the nanopores by setting a voltage across this membrane. If an analyte (e.g., a biopolymer such as DNA or RNA) passes through the pore or near its aperture, this event creates a characteristic disruption in current. The magnitude of the electric current density across a nanopore surface depends on the composition of DNA or RNA (e.g., the specific base) that is occupying the nanopore. Therefore, measurement of the current makes it possible to identify the sequence of the molecule in question.
Single molecule real-time (SMRT) sequencing (PacBio) is an exemplary single-molecule long- read sequencing technology that may be used to characterize libraries of engineered polyketide synthases or non-ribosomal peptide synthases that are prepared by any of the methods described herein. SMRT is a parallelized single molecule DNA sequencing method. SMRT utilizes a zero-mode waveguide (ZMW). A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
Examples
Example 1. Inactivation of a ketoreductase domain
A short peptide sequence from S303-KR6 was grafted onto X1 -KR6 using homology-based cloning. The catalytic Tyr is replaced with Phe (shown in red) and the active site aFG loop (show in blue) is also deleted (FIG. 5A). The resulting clone was conjugated into a Streptomyces expression host and fermented. Comparative LC-TOF analysis of unfractionated SPE samples of Compound 1 and C16-keto- Compound 1 indicated that the new compound had the desired M+H mass of 608.35 (FIG. 5B). We then performed Top Down mass spectrometry analysis by co-injecting purified native FKBP12 and Compound 1 or C16-keto-Compound 1 . This analysis again showed a mass difference of 2 consistent with the conversion at C16 of the hydroxyl to the ketone (FIG. 5C). C16-keto-Compound 1 was re-fermented at large scale, purified to homogeneity and the structure was confirmed by NMR spectroscopy. Example 2. Inactivation of dehydratase and enoyl reductase domains
Using the protocol from Example 1 , DH and ER domains in the PKS which produces Compound 1 were successfully deactivated as shown in FIG. 6A. Example 3. Inactivation of multiple domains simultaneously
Using the protocol from Example 1 , two domains were simultaneously deactivated as shown in
FIG. 6B.
Further, the expression profiles of E-06 (KR6*), a C16-keto-Compound 1 compound generated by inactivating the KR domain of module 6, and E36 (DH4*), a hydoxy-Compound 1 analog generated by inactivating the DH domain of module 4 were compared. When the validated KR and DH modifications were combined on a single construct, the resulting combinatorial compound E-74(KR6*-DH4*) produced the expected compound mass of 625.36 in good yield as detected by the Top-Down assay (FIG. 6c).
Example 4. Engineering of the Constant Region
The rapamycin/FK506 "constant region" is the conserved portion of the macrolide ring that binds
FKBP12. DH8 in the PKS which produces Compound 1 was inactivated by mutating the LPFXW motif to generate Compound 2 with a hydroxyl in the pyran ring of the constant region (FIG. 7A). The expected mass of 61 1 .38 (FIG. 7B) was observed by a Top-Down assay, which confirmed that Compound 2 retained FKBP12 binding affinity. The structure of the FKBP12: Compound 2:CEP250 complex (FIG. 7C) was solved by crystallization. The structure confirmed that (1 ) the FKBP12:CEP250 interface can accommodate the addition of the hydroxyl on the pyran ring, (2) the stereochemistry of the newly installed -OH group, and, as expected, (3) that CEP250 binding is also retained. The FKBP12-dependent CEP250 binding to Compound 2, as measured by TR-FRET, was increased as compared to Compound 2 (FIG. 7D).
The above data establish the utility of domain-level engineering to generate chemically-novel derivatives of PKS natural products which retaining biological function (i.e., target protein binding).
Example 5. Combinatorial domain engineering
An optimized Target-ID assay based on FKBP12 affinity enrichment and LC-MS/MS sequencing of tryptic peptides that allows for the identification of the protein targets of compounds in crude extracts was developed (FIG. 8A). Target-ID analysis confirmed that Compound 1 bound both CEP250 and CBY1 Ain 293T lysates, whereas Compound 3, a combinatorial compound derivative of Compound 1 , selectively bound CBY1 and not CEP250. The mass spectrometry-based Target-ID results were validated with TR-FRET data. The TR-FRET assay confirmed that CBY1 binds to Compound 1 (FIG. 8B). The data also confirmed that Compound 3 is specific for CBY1 and can no longer engage CEP250.
Furthermore, Compound 2 (FIG. 7A) is specific for CEP250 and not CBY1 . The structure of Compound 3 was confirmed by NMR, which indicated the successful inactivation of KR6, DH4 and ER5 domains via the domain-level engineering approach described above. Compound 2 also lacked the third carbonyl in the constant region, suggesting that CypB, the final tailoring step in Compound 1 biosynthesis, was unable to utilize Compound 2 as a productive substrate. The above data demonstrates that domain-level engineering results in compounds with
"reprogrammed" or altered target binding and therefore domain engineering can be utilized to generate molecules with new potential biological function. Example 6. Ring expansion resulting from ketoreductase deactivation
Each KR domain in modules 3-6 of the PKS which produces Compound 1 was systematically deactivated. Six sequences were tested for their ability to inactivate the KR domain (FIG. 9A).
Unexpectedly, a +44 mass in compound, Compound 4, was observed which was purified and the structure determined by NMR. The structure indicated that rather than installing a keto by inactivation of KR5, the ring size Compound 4 was expanded by 2 carbons, corresponding to an additional round of malonyl incorporation via PKS chain extension (FIG. 9B). The domain-level compound variant in the PKS which produces Compound 1 that yielded Compound 4 was a single Ala to Glu substitution in KR5 near the conserved catalytic YAAAN motif. This mutation may prevent access of the ketoreductase active site and, in doing so, may alter the kinetic balance between intramodular domain-domain handoff and intermodule transfer in the next cycle of polyketide elongation. This model predicts that module iteration is favored as a consequence of the KR5 mutation, which results in an additional malonyl incorporation event and expanded ring size.
Other Embodiments
It is to be understood that while the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and alterations are within the scope of the following claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above
Description, but rather is as set forth in the appended claims.
In the claims, articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
It is also noted that the term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is thus also encompassed and disclosed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any polynucleotide or protein encoded thereby; any method of production; any method of use) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

Claims

1 . An engineered polyketide synthase that comprises one or more modified domains having altered enzymatic activity relative to a reference polyketide comprising unmodified domains, wherein the engineered polyketide synthase is capable of producing a polyketide when expressed under conditions suitable to allow expression of a compound by the engineered polyketide synthase.
2. The engineered polyketide synthase of claim 1 , wherein the polyketide synthase comprises two or more modified domains having altered enzymatic activity.
3. The engineered polyketide synthase of claim 1 or 2, wherein at least one modified domain has decreased enzymatic activity.
4. The engineered polyketide synthase of claim 3, wherein at least one modified domain is functionally inactive.
5. The engineered polyketide synthase of any one of claims 1 to 4, wherein the modified domain is a β-ketone processing domain.
6. The engineered polyketide synthase of claim 5, wherein the β-ketone processing domain is a ketoreductase, a dehydratase, or an enoylreductase.
7. The engineered polyketide synthase of claim 6, wherein the β-ketone processing domain comprises a portion having at least 90% sequence identity to the conserved region of any one of SEQ ID NO: 1 -9.
8. The engineered polyketide synthase of claim 6, wherein the β-ketone processing domain is a ketoreductase, wherein the ketoreductase (a) comprises an amino acid other than tyrosine at the position corresponding to the tyrosine in the conserved YAAAN catalytic motif and does not comprise the conserved aFG helix in SEQ ID NO: 1 ; (b) comprises a glutamic acid residue at the position
corresponding to alanine 6632 of S9-pksA ORF (the change in S9) in SEQ ID NO: 2; or (c) does not comprise the amino acids corresponding to amino acids 3386 to 3516 of WT S12-pksB ORF of SEQ ID NO: 3.
9. The engineered polyketide synthase of claim 6, wherein the β-ketone processing domain is a dehydratase, wherein the dehydratase comprises (a) an aspartic acid at the position corresponding to the glycine at position 4288 in pksB of S679-pksB ORF in the conserved HXXXGXXXXP motif of SEQ ID NO: 4; (b) a substitution in the conserved LPFXW motif at the position corresponding to position 3066 to 3070 in S12-pksB ORF in SEQ ID NO: 5; (c) a deletion between Pro 6844 and Trp 6874 of S679-pksA ORF of SEQ ID NO: 6; or (d) a substitution or deletion at the positions corresponding to A, B, C, and D of SEQ ID NO: 7.
10. The engineered polyketide synthase of claim 6, wherein the β-ketone processing domain is an enoylreductase, wherein the enoylreductase does not comprise a lysine at the position corresponding to position 1546 of S12-pksB ORF in SEQ ID NO: 8 and/or the aspartic acid at the position corresponding to position 1568 of S12-pksB in SEQ ID NO: 8 or SEQ ID NO: 9.
1 1 . A polyketide synthase comprising:
(a) a first domain comprising a conserved region of a domain of a first polyketide synthase; and
(b) a second domain comprising a conserved region of a domain of a second polyketide synthase.
12. The polyketide synthase of claim 1 1 , wherein at least one of the first domain and the second domain is a β-ketone processing domain.
13. The polyketide synthase of claim 1 1 or 12, wherein the first domain and the second domain are both β-ketone processing domains.
14. The polyketide synthase of claim 12 or 13, wherein the β-ketone processing domain is a ketoreductase, a dehydratase, or an enoylreductase.
15. The polyketide synthase of any one of claims 1 1 to 14, wherein at least one of the first domain and the second domain is a functionally inactive domain.
16. The polyketide synthase of claim 15, wherein both of the first domain and the second domain are functionally inactive domains.
17. The polyketide synthase of any one of claims 1 1 to 16, wherein the polyketide synthase comprises (c) a conserved region of a domain of a third polyketide synthase or the conserved region of a second domain of the second polyketide synthase.
18. The polyketide synthase of claim 17, wherein the third domain is a functionally inactive domain.
19. The polyketide synthase of any one of claims 1 1 to 18, wherein the polyketide synthase comprises (d) a conserved region of a domain of a fourth polyketide synthase, the conserved region of a second domain of the third polyketide synthase, or the conserved region of a third domain of the second polyketide synthase.
20. The polyketide synthase of claim 19, wherein the fourth domain is functionally inactive.
21 . The polyketide synthase of any one of claims claim 15 to 20, wherein the functionally inactive domain comprises the amino acid sequence of the conserved region of any one of SEQ ID NO: 1 -9.
22. A chimeric polyketide synthase, wherein at least one domain of the polyketide synthase has been modified as compared to a polyketide synthase having the sequence of SEQ ID NO: 10 or 1 1 , wherein the modification results in altered enzymatic activity.
23. A chimeric polyketide synthase, wherein at least one ketoreductase domain (a) comprises an amino acid other than tyrosine at the position corresponding to the tyrosine in the conserved YAAAN catalytic motif and does not comprise the conserved aFG helix in SEQ ID NO: 1 ; (b) comprises a glutamic acid residue at the position corresponding to alanine 6632 of S9-pksA ORF in SEQ ID NO: 2; or (c) does not comprise the amino acids corresponding to amino acids 3386 to 3516 of WT S12-pksB ORF of SEQ ID NO: 3.
24. A chimeric polyketide synthase, wherein at least one dehydratase domain (a) comprises an aspartic acid at the position corresponding to the glycine at position 4288 in pksB of S679-pksB ORF in the conserved HXXXGXXXXP motif of SEQ ID NO: 4; (b) comprises a substitution in the conserved LPFXW motif at the position corresponding to position 3066 to 3070 in S12-pksB ORF in SEQ ID NO: 5; (c) comprises a deletion corresponding to positions between Pro 6844 and Trp 6874 of S679-pksA ORF of SEQ ID NO: 6; or (d) comprises a substitution or deletion at the positions corresponding to A, B, C, and D of SEQ ID NO: 7.
25. A chimeric polyketide synthase, wherein at least one enoylreductase domain does not comprise a lysine at the position corresponding to position 1546 of S12-pksB ORF in SEQ ID NO: 8 and/or the aspartic acid at the position corresponding to position 1 568 of S12-pksB in SEQ ID NO: 8 or 9.
26. A chimeric polyketide synthase comprising a domain having at least 80% sequence identity to the amino acid sequence of (a) SEQ ID NO: 7, 8, or 9; (b) SEQ ID NO: 10, 1 1 , or 12; (c) SEQ ID NO: 13, 14, or 15; (d) SEQ ID NO: 16, 17, or 18; (e) SEQ ID NO: 19, 20, 21 , or 22; (f) SEQ ID NO: 23, 24, 25, or 26; (g) SEQ ID NO: 27, 28, 29, or 30; or (h) SEQ ID NO: 31 or 32.
27. A nucleic acid encoding a polyketide synthase of any one of claims 1 to 26.
28. The nucleic acid of claim 27, wherein the nucleic acid further encodes an LAL, wherein the sequence encoding the LAL is operatively linked to the sequence encoding the polyketide synthase.
29. The nucleic acid of claim 28, wherein the LAL is a heterologous LAL.
30. The nucleic acid of claim 28 or 29, wherein LAL comprises a portion having at least 80% identity to SEQ ID NO: 38.
31 . The nucleic acid of claim 30, wherein the LAL comprises a portion having the sequence of SEQ ID NO: 38.
32. The nucleic acid of claim 31 , wherein the LAL has the sequence of SEQ ID NO: 38.
33. The nucleic acid of any one of claims 28 to 32, wherein the nucleic acid encoding the LAL lacks a TTA inhibitory codon in an open reading frame.
34. The nucleic acid of any one of claims 27 to 33, wherein the nucleic acid further comprises an LAL binding site, wherein the sequence encoding the LAL binding site is operatively linked to the sequence encoding the polyketide synthase.
35. The nucleic acid of claim 34, wherein the LAL binding site comprises a portion having at least 80% sequence identity to the sequence of SEQ ID NO: 39.
36. The nucleic acid of claim 35, wherein the LAL binding site comprises a portion having the sequence of SEQ ID NO: 39.
37. The nucleic acid of claim 36, wherein the LAL binding site has of the sequence of SEQ ID
NO: 39.
38. The nucleic acid of claim 34, wherein the LAL binding site has the sequence GGGGGT (SEQ ID NO: 40).
39. The nucleic acid of any one of claims 34 to 38, wherein the binding of an LAL to the LAL binding site promotes expression of the polyketide synthase.
40. The nucleic acid of any one of claims 27 to 39, wherein the nucleic acid further encodes a nonribosomal peptide synthase.
41 . The nucleic acid of any one of claims 27 to 40, wherein the nucleic acid further encodes a first P450 enzyme.
42. The nucleic acid of claim 41 , wherein the nucleic acid further encodes a second P450 enzyme.
43. An expression vector comprising a nucleic acid of any one of claims 27 to 42.
44. The expression vector of claim 43, wherein the expression vector is an artificial chromosome.
45. The expression vector of claim 44, wherein the artificial chromosome is a bacterial artificial chromosome.
46. A host cell comprising an expression vector of any one of claims 43 to 45.
47. A host cell comprising a polyketide synthase of any one of claims 1 to 26, wherein the polyketide is heterologous to the host cell.
48. The host cell of claim 46 or 47, wherein the host cell naturally lacks an LAL.
49. The host cell of any one of claims 46 to 48, wherein the host cell naturally lacks an LAL binding site.
50. The host cell of any one of claims 46 to 49, wherein the host cell comprises an LAL capable of binding to an LAL binding site and regulating expression of a polyketide synthase.
51 . The host cell of claim 50, wherein the LAL is heterologous.
52. The host cell of claim 50 or 51 , wherein the LAL comprises a portion having at least 80% identity to the sequence of SEQ ID NO: 38.
53. The host cell of any one of claims 46 to 52, wherein the host cell is a bacterium.
54. The host cell of claim 53, wherein the bacterium is an actinobacterium.
55. The host cell of claim 54, wherein the actinobacterium is Streptomyces ambofaciens, Streptomyces hygroscopicus, or Streptomyces malayensis.
56. The host cell of claim 55, wherein the actinobaceterium is S1391 , S1496, or S2441 .
57. The host cell of any one of claims 46 to 56, wherein the host cell has been modified to enhance expression of a polyketide synthase.
58. The host cell of claim 57, wherein the host cell has been modified to enhance expression of a compound-producing protein by (i) deletion of an endogenous gene cluster which expresses a compound-producing protein; (ii) insertion of a heterologous gene cluster which expresses a compound- producing protein; (iii) exposure of the host cell to an antibiotic challenge; and/or (iv) introduction of a heterologous promoter that results in an at least 2-fold increase in expression of a compound compared to the homologous promoter.
59. A method of producing a polyketide, the method comprising culturing a host cell of any one of claims 46 to 58 under suitable conditions.
60. A method of producing a polyketide, the method comprising culturing a host cell engineered to express a polyketide synthase of any one of claims 1 to 26 under conditions suitable for polyketide synthase to produce a polyketide.
61 . A method of modulating the activity of a polyketide synthase, the method comprising:
(a) providing a parent nucleic acid sequence encoding a parent polyketide synthase; and
(b) modifying at least one codon of the parent nucleic acid sequence, wherein the codon specifies a residue in a conserved motif of at least one domain of the parent polyketide synthase; and
wherein the modification results in an alteration of an enzymatic or regulatory activity of the at least one domain.
62. A method of producing a compound, the method comprising:
(a) providing a parent nucleic acid encoding a parent polyketide synthase;
(b) modifying at least one codon of the parent nucleic acid to create a modified nucleic acid encoding a modified polyketide synthase capable of producing a compound, wherein the codon specifies a residue in a conserved domain of at least one domain of the polyketide synthase and wherein the modification results in an alteration of the enzymatic activity of the at least one domain of the polyketide synthase;
(c) introducing the modified nucleic acid to a host cell; and
(d) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase;
thereby producing a compound.
63. A method of producing a compound, the method comprising:
(a) providing a parent polyketide synthase capable of producing a compound;
(b) determining the amino acid sequence of the parent polyketide synthase;
(c) providing a parent nucleic acid encoding the parent polyketide synthase;
(d) modifying at least one codon of the parent nucleic acid to create a modified nucleic acid sequence encoding a modified polyketide synthase capable of producing a compound, wherein the codon specifies a residue in a conserved domain of at least one domain of the polyketide synthase and wherein the modification results in an alteration of the enzymatic activity of the at least one domain;
(e) introducing the modified nucleic acid to a host cell;
(f) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase; and
(g) recovering the compound produced by the modified polyketide synthase;
thereby producing a compound.
64. A method of producing a compound, the method of comprising: (a) determining the structure of a parent polyketide synthase;
(b) producing a parent nucleic acid encoding the parent polyketide synthase;
(c) modifying the nucleic acid to produce a modified nucleic acid encoding a modified polyketide synthase, wherein at least one domain of the modified polyketide synthase has altered enzymatic activity compared to the parent polyketide synthase;
(d) introducing the modified nucleic acid sequence to a host cell; and
(e) culturing the host cell under conditions suitable to allow expression of a compound by the modified polyketide synthase;
thereby producing a compound.
65. A method of producing a library of compounds, the method comprising:
(a) providing a parent nucleic acid sequence encoding a parent polyketide synthase;
(b) modifying at least one codon of the parent nucleic acid sequence to create a first modified nucleic acid encoding a first modified polyketide synthase capable of producing a compound;
(c) modifying at least one codon of the parent nucleic acid to create a second modified nucleic acid encoding a second modified polyketide synthase capable of producing a compound, wherein the first and second modified nucleic acids are different;
(d) introducing the first and the second modified nucleic acid sequences to one or more host cells; and
(e) culturing the one or more host cells under conditions suitable to allow expression of a compound by the first and the second modified polyketide synthase;
thereby producing a library of compounds.
66. A compound produced by the method of any one of claims 59 to 65.
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